DNA encoding interleukin-4 receptors

ABSTRACT

Mammalian Interleukin-4 receptor proteins, DNAs and expression vectors encoding mammalian IL-4 receptors, and processes for producing mammalian IL-4 receptors as products of cell culture, are disclosed. A method for suppressing an IL-4-dependent immune or inflammatory response in a mammal, including a human, by administering an effective amount of soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier.

BACKGROUND OF THE INVENTION

This application is a continuation of application Ser. No. 08/094,669,filed Jul. 20, 1993, now U.S. Pat. No. 5,599,905, which is a divisionalof application Ser. No. 07/480,694, filed Feb. 14, 1990, currentlypending, which is a continuation-in-part of application Ser. No.07/370,924, filed Jun. 23, 1989, now abandoned which is acontinuation-in-part of Ser. No. 07/326,156, filed Mar. 20, 1989, nowabandoned, which is a continuation-in-part of Ser. No. 07/319,438, filedMar. 2, 1989, now abandoned, which is a continuation-in-part of Ser. No.07/265,047, filed Oct. 31, 1988, now abandoned.

The present invention relates generally to cytokine receptors and, morespecifically, to Interleukin-4 receptors.

Interleukin-4 (IL-4, also known as B cell stimulating factor, or BSF-1)was originally characterized by its ability to stimulate theproliferation of B cells in response to low concentrations of antibodiesdirected to surface immunoglobulin. More recently, IL-4 has been shownto possess a far broader spectrum of biological activities, includinggrowth co-stimulation of T cells, mast cells, granulocytes,megakaryocytes, and erythrocytes. In addition, IL-4 stimulates theproliferation of several IL-2- and IL-3-dependent cell lines, inducesthe expression of class II major histocompatibility complex molecules onresting B cells, and enhances the secretion of IgE and IgG1 isotypes bystimulated B cells. Both murine and human IL-4 have been definitivelycharacterized by recombinant DNA technology and by purification tohomogeneity of the natural murine protein (Yokota et al., Proc. Natl.Acad. Sci. USA 83:5894, 1986; Noma et al., Nature 319:640, 1986; andGrabstein et al., J. Exp. Med. 163:1405, 1986).

The biological activities of IL-4 are mediated by specific cell surfacereceptors for IL-4 which are expressed on primary cells and in vitrocell lines of mammalian origin. IL-4 binds to the receptor, which thentransduces a biological signal to various immune effector cells.Purified IL-4 receptor (IL-4R) compositions will therefore be useful indiagnostic assays for IL-4 or IL-4 receptor, and in raising antibodiesto IL-4 receptor for use in diagnosis or therapy. In addition, purifiedIL-4 receptor compositions may be used directly in therapy to bind orscavenge IL-4, providing a means for regulating the biologicalactivities of this cytokine.

Although IL-4 has been extensively characterized, little progress hasbeen made in characterizing its receptor. Numerous studies documentingthe existence of an IL-4 receptor on a wide range of cell types havebeen published; however, structural characterization has been limited toestimates of the molecular weight of the protein as determined bySDS-PAGE analysis of covalent complexes formed by chemical cross-linkingbetween the receptor and radiolabeled IL-4 molecules. Ohara et al.(Nature 325:537, 1987) and Park et al. (Proc. Natl. Acad. Sci. USA84:1669, 1987) first established the presence of an IL-4 receptor usingradioiodinated recombinant murine IL-4 to bind a high affinity receptorexpressed in low numbers on B and T lymphocytes and a wide range ofcells of the hematopoietic lineage. By affinity cross-linking ¹²⁵ I-IL-4to IL-4R, Ohara et al. and Park et al. identified receptor proteinshaving apparent molecular weights of 60,000 and 75,000 daltons,respectively. It is possible that the small receptor size observed onthe murine cells represents a proteolytically cleaved fragment of thenative receptor. Subsequent experiments by Park et al. (J. Exp. Med.166.476, 1987) using yeast derived recombinant human IL-4 radiolabeledwith ¹²⁵ I showed that human IL-4 receptor is present not only on celllines of B, T, and hematopoietic cell lineages, but is also found onhuman fibroblasts and cells of epithelial and endothelial origin. IL-4receptors have since been shown to be present on other cell lines,including CBANN splenic B cells (Nakajima et al., J. Immunol. 139:774,1987), Burkitt lymphoma Jijoye cells (Cabrillat et al., Biochem. &Biophys. Res. Commun. 149:995, 1987), a wide variety of hemopoietic andnonhemopoietic cells (Lowenthal et al., J. Immunol. 140:456, 1988), andmurine Lyt-2-L3T4- thymocytes. More recently, Park et al. (UCLASymposia, J. Cell Biol. Suppl. 12A, 1988) reported that, in the presenceof sufficient protease inhibitors, ¹²⁵ I-IL-4-binding plasma membranereceptors of 138-145 kDa could be identified on several murine celllines. Considerable controversy thus remains regarding the actual sizeand structure of IL-4 receptors.

Further study of the structure and biological characterstics of IL-4receptors and the role played by IL-4 receptors in the responses ofvarious cell populations to IL-4 or other cytokine stimulation, or ofthe methods of using IL-4 receptors effectively in therapy, diagnosis,or assay, has not been possible because of the difficulty in obtainingsufficient quantities of purified IL-4 receptor. No cell lines havepreviously been known to express high levels of IL-4 receptorsconstitutively and continuously, and in cell lines known to expressdetectable levels of IL-4 receptor, the level of expression is generallylimited to less than about 2000 receptors per cell. Thus, efforts topurify the IL-4 receptor molecule for use in biochemical analysis or toclone and express mammalian genes encoding IL-4 receptor have beenimpeded by lack of purified receptor and a suitable source of receptormRNA.

SUMMARY OF THE INVENTION

The present invention provides DNA sequences encoding mammalianInterleukin-4 receptors (IL-4R) or subunits thereof. Preferably, suchDNA sequences are selected from the group consisting of: (a) CDNA cloneshaving a nucleotide sequence derived from the coding region of a nativeIL-4R gene; (b) DNA sequences capable of hybridization to the CDNAclones of (a) under moderately stringent conditions and which encodebiologically active IL-4R molecules; and (c) DNA sequences which aredegenerate, as a result of the genetic code, to the DNA sequencesdefined in (a) and (b) and which encode biologically active IL-4 Rmolecules. The present invention also provides recombinant expressionvectors comprising the DNA sequences defined above, recombinant IL-4Rmolecules produced using the recombinant expression vectors, andprocesses for producing the recombinant IL-4R molecules using theexpression vectors.

The present invention also provides substantially homogeneous proteincompositions comprising mammalian IL-4R. The full length murine moleculeis a glycoprotein having a molecular weight of about 130,000 to about140,000 M_(r) by SDS-PAGE. The apparent binding affinity (K_(a)) for COScells transfected with murine IL-4 receptor clones 16 and 18 from theCTLL 19.4 library is 1 to 8×10⁹ M⁻¹. The K_(a) for COS cells transfectedwith murine IL-4 receptor clones 7B9-2 and 7B9-4 from the murine 7B9library is 2×10⁹ to 1×10¹⁰ M⁻¹. The mature murine IL-4 receptor moleculehas an N-terminal amino acid sequence as follows: IKVLGEPTCFSDYIRTSTCEW.

The human IL-4R molecule is believed to have a molecular weight ofbetween about 110,000 and 150,000 M_(r) and has an N-terminal amino addsequence, predicted from the cDNA sequence and by analogy to thebiochemically determined N-terminal sequence of the mature murineprotein, as follows: MKVLQEPTCVSDYMSISTCEW.

The present invention also provides compositions for use in therapy,diagnosis, assay of IL-4 receptor, or in raising antibodies to IL-4receptors, comprising effective quantities of soluble receptor proteinsprepared according to the foregoing processes. Such soluble recombinantreceptor molecules include truncated proteins wherein regions of thereceptor molecule not required for IL-4 binding have been deleted.

The present invention also provides a method for suppressing IL-4mediated immune or inflammatory responses. This method comprisesadministering an effective quantity of soluble IL-4 receptor (sIL-4R),in association with a pharmaceutical carrier, to a mammal, includingman. sIL-4R suppresses IL-4 dependent immune or inflammatory responses,including, for example, B cell mediated activities, such as B cellproliferation, immunoglobulin secretion, and expression of FcεR whichare induced by IL-4. sIL-4R also suppresses cytotoxic T cell induction.Clinical applications of sIL-4R include, for example, use in allergytherapy to selectively suppress IgE synthesis and use in transplantationtherapy to prevent allograft rejection. sIL-4R is also useful tosuppress delayed-type hypersensitivity or contact hypersensitivityreactions. sIL-4R is highly specific in its immunosuppressive activitybecause it suppresses only IL-4 mediated immune responses.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows restriction maps of cDNA clones containing the codingregions (denoted by a bar) of the murine and human IL-4R cDNAs. Therestriction sites EcoRI, PvuII, Hinc II and Sst I are represented by theletters R, P, H and S, respectively.

FIGS. 2A-C depict the cDNA sequence and the derived amino acid sequenceof the coding region of a murine IL-4 receptor, as derived from clone7B9-2 of the 7B9 library. The N-terminal isoleucine of the matureprotein is designated amino acid number 1. The coding region of thefull-length membrane-bound protein from clone 7B9-2 is defined by aminoacids 1-785. The ATC codon specifying the isoleucine residueconstituting the mature N-terminus is underlined at position 1 of theprotein sequence; the putative transmembrane region at amino acids209-232 is also underlined. The sequences of the coding regions ofclones 7B9-4 and clones CTLL-18 and CTLL-16 of the CTLL 19.4 library areidentical to that of 7B9-2 except as follows. The coding region ofCTLL-16 encodes a membrane-bound IL-4-binding receptor defined by aminoacids -25 through 233 (including the putative 25 amino acid signalpeptide sequence), but is followed by a TAG terminator codon (not shown)which ends the open reading frame. The nucleic acid sequence indicatesthe presence of a splice donor site a this position C indicated by anarrow in FIG. 1) and a splice acceptor site near the 3' end (indicatedby a second arrow), suggesting that CTLL-16 was derived from anunspliced MRNA intermediate. Clones 7B94 and CTLL-18 encode amino acids23 through 199 and 25 through 199, respectively. After amino acid 199, a114-base pair insert (identical in both clones and shown by an open boxin FIG. 1) introduces six new amino acids, followed by a terminationcodon. This form of the receptor is soluble.

FIG. 3 is a schematic illustration of the mammalian high expressionplasmid pCAV/NOT, which is described in greater detail in Example 8.

FIGS. 4A-C depict the coding sequence of a human IL-4 receptor cDNA fromclone T22-8, which was obtained from a CDNA library derived from the Tcell line T22. The predicted N-terminal methionine of the mature proteinand the transmembrane region are underlined. FIGS. 5A-B are a comparisonof the predicted amino acid sequences of human (top line) and murine(bottom line) IL-4 receptor cDNA clones.

FIG. 6 shows the inhibition of B cell proliferation with IL-4 (panel A)or IL-1 (panel B) at various doses either alone (∘) or in the presenceof sIL-4 (□), sIL-1(▪) or anti-IL-4 antibody () as described in Example15.

FIG. 7 shows the inhibition of B cell proliferation with fixedconcentrations of 10 (), 1 (□), 0.1 (∘) or 0 (Δ) ng/ml of IL-4 (panelsA-C) and IL-1 (panels D-F) at various doses of sIL-4R (panels A & D),anti-IL-4 antibody (panels B & E) and sIL-1 R (panels C & F) asdescribed in Example 15.

FIG. 8 shows the inhibition of immunoglobulin class switching withvarious doses of IL-4 and with sIL-4R (□), sIL-1R (▪), or anti-IL-4antibody () or medium control (Δ) as described in Example 16.

FIG. 9 shows the inhibition of IL-4-induced immunoglobulin classswitching with fixed concentration of IL-4 and various doses of sIL-4R(□), sIL-1R (▪), anti-IL-4 antibody () or medium control (Δ) asdescribed in Example 16.

FIG. 10 shows the inhibition of MHC class II antigen expression with(dashed line) or without (solid line) IL-4 in the presence of mediumcontrol (panel A), sIL-4R (panel B), anti-IL-4 antibody (panel C) orsIL-1R (panel D) as described in Example 17.

FIG. 11 shows the inhibition of FcεR (CD23) expression with (dashedline) or without (solid line) IL-4 in the presence of medium control(panel A), sIL-4 R (panel B), anti-IL-4 antibody (panel C) or sIL-1R(panel D) as described in Example 17.

FIG. 12 shows the inhibition of antigen specific polyclonal IgE levelsby sIL-4R as described in Example 18.

FIG. 13 shows the inhibition of antigen specific anti-TNP-KLH IgE levelsby sIL-4R as described in Example 18.

FIG. 14 shows that sIL-4R doses of 1, 5 or 25 ug on days -1, 0 and +1,as described in Example 19, do not significantly inhibit antigenspecific anti-IgD IgE levels.

FIG. 15 shows the inhibition of contact hypersensitivity responses toDNFB with sIL-4R as described in Example 20.

FIG. 16 shows the inhibition of delayed-type hypersensitivity responsesto SRBC with sIL-4R as described in Example 21.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Interleukin-4" and "IL-4 " (also referred to as B cell stimulatingfactor, or BSF-1) is a T cell-derived cytokine involved in theregulation of immune and inflammatory responses. The biologicalactivities of IL-4 are mediated through binding to specific cell surfacereceptors, referred to as "Interleukin-4 receptors", IL-4 receptors orsimply "IL-4 R". "IL- mediated" immune or inflammatory responses includeall biological responses which are caused by the binding of IL-4 to anative IL-4 receptor (bound to a cell surface) or which may be inhibitedor suppressed by preventing IL-4 from binding to a native IL-4 receptor.IL-4 mediated biological responses include, for example, IL-4 inducedproliferation of antigen-primed B lymphocytes, expression of class IImajor histocompatibility complex molecules on resting B cells, secretionand expression of antibodies of the IgE and IgG1 isotype, and regulationof the expression of the low affinity Fc receptor for IgE (CD23) onlymphocytes and monocytes. Outside the B lymphocyte compartment, IL-4mediated biological responses include the proliferation of a variety ofprimary cells and in vitro cell lines, including factor-dependent T celland mast cell lines, murine and human T lymphocytes, thymocytes, andconnective tissue-type mast cells. IL-4 also induces both murine andhuman cytotoxic T cells. Under certain conditions, IL-4 inhibits theresponse of lymphoid cells to IL-2. IL-4 acts on both murine and humanhematopoietic progenitor cells to either stimulate or suppress in vitroformation of colonies in combination with known colony stimulatingfactors. IL-4 also induces class I and class II MHC molecules on moremature cells of the monocytic lineage, enhances antigen presentingability and promotes formation of giant multinucleated cells. Specificclinical conditions which may be mediated by IL-4 include, for example,graft rejection, graft versus host disease, allergy, asthma anddelayed-type hypersensitivity responses.

As used herein, the terms "IL-4 receptor" or "IL-4 R" refer to proteinswhich bind interleukin-4 (IL-4) molecules and, in their nativeconfiguration as intact human plasma membrane proteins, play a role intransducing the biological signal provided by IL-4 to a cell. Intactreceptor proteins generally include an extracellular region which bindsto a ligand, a hydrophobic transmembrane region which causes the proteinto be immobilized within the plasma membrane lipid bilayer, and acytoplasmic or intracellular region which interacts with cytoplasmicproteins and/or chemicals to deliver a biological signal to effectorcells via a cascade of chemical reactions within the cytoplasm of thecell. The hydrophobic transmembrane region and a highly charged sequenceof amino acids in the cytoplasmic region immediately following thetransmembrane region cooperatively function to haft transport of theIL-4 receptor across the plasma membrane.

"IL-4 receptors" are proteins having amino acid sequences which aresubstantially similar to the native mammalian Interleukin-4 receptoramino acid sequences disclosed in FIGS. 2 and 4 or fragments thereof,and which are biologically active as defined below, in that they arecapable of binding Interleukin-4 (IL-4) molecules or transducing abiological signal initiated by an IL-4 molecule binding to a cell, orcross-reacting with anti-IL-4R antibodies raised against IL-4R fromnatural (i.e., nonrecombinant) sources. The native human IL-4 receptormolecule has an apparent molecular weight by SDS-PAGE of about 140kilodaltons (kDa). The native murine IL-4 receptor molecule has anapparent molecular weight by SDS-PAGE of about 140 kilodaltons (kDa).The terms "IL-4 receptor" or "IL-4R" include, but are not limited to,soluble IL-4 receptors, as defined below. Specific IL-4 receptorpolypeptides are designated herein by parenthetically indicating theamino acid sequence numbers, followed by any additional amino acidsequences. For Example, human IL-4R (1-207) refers to a human IL-4Rprotein having the sequence of amino acids 1-207 as shown in FIG. 4A.Human IL-4R (1-184) Pro Ser Asn Glu Asn refers to a human IL-4R proteinhaving the sequence of amino acids 1-184 as shown in FIG. 4A, followedby the amino acid sequence Pro Ser Asn Glu Asn. As used throughout thespecification, the term "mature" means a protein expressed in a formlacking a leader sequence as may be present in full-length transcriptsof a native gene. Various bioequivalent protein and amino acid analogsare described in the detailed description of the invention.

"Substantially similar" IL-4 receptors include those whose amino acid ornucleic acid sequences vary from the native sequences by one or moresubstitutions, deletions, or additions, the net effect of which is toretain biological activity of the IL-4R protein. For example, nucleicacid subunits and analogs are substantially similar to the specific DNAsequences disclosed herein if: (a) the DNA sequence is derived from thecoding region of a native mammalian IL-4R gene; (b) the DNA sequence iscapable of hybridization to DNA sequences of (a) under moderatelystringent conditions and which encode biologically active IL-4Rmolecules; or DNA sequences which are degenerate as a result of thegenetic code to the DNA sequences defined in (a) or (b) and which encodebiologically active IL-4R molecules. Substantially similar analogproteins will generally be greater than about 30 percent similar to thecorresponding sequence of the native IL-4 R. Sequences having lesserdegrees of similarity but comparable biological activity are consideredto be equivalents. More preferably, the analog proteins will be greaterthan about 80 percent similar to the corresponding sequence of thenative IL-4R, in which case they are defined as being "substantiallyidentical." In defining nucleic acid sequences, all subject nucleic acidsequences capable of encoding substantially similar amino acid sequencesare considered substantially similar to a reference nucleic acidsequence. Percent similarity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0, available from the University of Wisconsin Genetics Computer Grup(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) which are similar, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, ed., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

"Soluble IL-4 receptor" or "slL4-R" as used in the context of thepresent invention refers to a protein, or a substantially equivalentanalog, having an amino acid sequence corresponding to the extracellularregion of native IL-4 receptors, for example, polypeptides having theamino acid sequences substantially equivalent to the sequences of aminoacids 1-208 of FIG. 2, amino acids 1-207 of FIG. 4 or to the amino acidsequences discussed in Examples 8C and 9. Equivalent sIL-4Rs includepolypeptides which vary from the sequences shown in FIGS. 2 or 4 by oneor more substitutions, deletions, or additions, and which retain theability to bind IL-4 and inhibit the ability of IL-4 to transduce asignal via cell surface bound IL-4 receptor proteins. Because sIL-4Rproteins are devoid of a transmembrane region, they are secreted fromthe host cell in which they are produced. When administered intherapeutic formulations, sIL-4R proteins circulate in the body and bindto circulating IL-4 molecules, preventing interaction of IL-4 withnatural IL-4 receptors and inhibiting transduction of IL-4 mediatedbiological signals, such as immune or inflammatory responses. Theability of a polypeptide to inhibit IL-4 signal transduction can bedetermined by transfecting cells with recombinant IL-4 receptor DNAs toobtain recombinant receptor expression. The cells are then contactedwith IL-4 and the resulting metabolic effects examined. If an effectresults which is attributable to the action of the ligand, then therecombinant receptor has signal transducing activity. Exemplaryprocedures for determining whether a polypeptide has signal transducingactivity are disclosed by ldzerda et al., J. Exp. Med., March 1990 inpress, Curtis et al., Proc. NatL. Acad. Sci. USA 86.3045 (1989), Pryweset al., EMBO J. 5:2179 (1986) and Chou et al., J. Biol. Chem. 262:1842(1987). Alternatively primary cells of cell lines which express anendogenous IL-4 receptor and have a detectable biological response toIL-4 could also be utilized. Such is the case with the CTLL-2 cell linewhich responds by short term proliferation in response to either IL-2 orIL-4; the IL-4 induced proliferation can be blocked specifically by theaddition of exogenous soluble IL-4 R (Mosley et al., Cell 59:335 (1989).In addition, any one of the in vivo or in vitro assays described inExamples 14-23 can be utilized to determine whether a soluble IL-4Rinhibits transduction of a specific IL-4 mediated biological signal. Thecloning, sequencing and expression of full-length and soluble forms ofthe receptor for murine IL-4 have recently been described by theapplicants, Mosley et al., Cell 59:335, 1989, which publication isincorporated herein by reference.

"Recombinant," as used herein, means that a protein is derived fromrecombinant (e.g., microbial or mammalian) expression systems. "Mobia"refers to recombinant proteins made in bacterial or fungal (e.g., yeast)expression systems. As a product, "recombinant microbial" defines aprotein produced in a microbial expression system which is essentiallyfree of native endogenous substances. Protein expressed in mostbacterial cultures, e.g., E. coli, will be free of glycan. Proteinexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells. "Biolgically active," as used throughoutthe specification as a characteristic of IL-4 receptors, means that aparticular molecule shares sufficient amino acid sequence similaritywith the embodiments of the present invention disclosed herein to becapable of binding detectable quantities of IL-4, transducing an IL-4signal to a cell, for example, as a component of a hybrid receptorconstruct, or cross-reacting with anti-IL-4R antibodies raised againstIL-4R from natural (i.e., nonrecombinant) sources. Preferably,biologically active IL-4 receptors within the scope of the presentinvention are capable of binding greater than 0.1 nmoles IL-4 per nmolereceptor, and most preferably, greater than 0.5 nmole IL-4 per nmolereceptor in standard binding assays (see below).

"DNA sequence" refers to a DNA molecule, in the form of a separatefragment or as a component of a larger DNA construct, which has beenderived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. Genomic DNA containing the relevantsequences could also be used. Sequences of non-translated DNA may bepresent 5' or 3' from the open reading frame, where the same do notinterfere with manipulation or expression of the coding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.DNA sequences encoding the proteins provided by this invention can beassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a replicable DNA constructused either to amplify or to express DNA which encodes IL-4R and whichincludes a transcriptional unit comprising an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription and translation initiation and terminationsequences. Structural elements intended for use in yeast expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it may include an N-terminal methionine residue. This residue mayoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

"Recombinant microbial expression system" means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E coli or yeast such as S. cerevisiae, which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Proteins and Analogs

The present invention provides substantially homogeneous recombinantmammalian IL-4R polypeptides substantially free of contaminatingendogenous materials and, optionally, without associated native-patternglycosylation. The native murine and human IL-4 receptor molecules arerecovered from cell lysates as glycoproteins having an apparentmolecular weight by SDS-PAGE of about 130-145 kilodaltons (kDa).Mammalian IL-4R of the present invention includes, by way of example,primate, human, murine, canine, feline, bovine, ovine, equine andporcine IL-4R. Derivatives of IL-4R within the scope of the inventionalso include various structural forms of the primary protein whichretain biological activity. Due to the presence of ionizable amino andcarboxyl groups, for example, an IL-4R protein may be in the form ofacidic or basic salts, or in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Covalent derivatives are prepared bylinking particular functional groups to IL-4R amino acid side chains orat the N- or C-termini. Other derivatives of IL-4R within the scope ofthis invention include covalent or aggregative conjugates of IL-4R orits fragments with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. For example,the conjugated peptide may be a signal (or leaded) polypeptide sequenceat the N-terminal region of the protein which co-translationally orpost-translationally directs transfer of the protein from its site ofsynthesis to its site of function inside or A is outside of the cellmembrane or wall (e.g., the yeast α-factor leader). IL-4R proteinfusions can comprise peptides added to facilitate purification oridentification of IL-4R (e.g., poly-His). Specific examples of apoly-HIS fusion construct that is biologically active are soluble humanIL-4 R (1-207) His His and soluble human IL-4R (1-207) His His His HisHis His. The amino acid sequence of IL-4 receptor can also be linked tothe peptide Asp-Tyr-Lys-AspAsp-AspAsp-Lys (DYKDDDDK) (Hopp et al.,Bio/Technology 6:1204, 1988.) The latter sequence is highly antigenicand provides an epitope reversibly bound by a specific monoclonalantibody, enabling rapid assay and facile purification of expressedrecombinant protein. This sequence is also specifically cleaved bybovine mucosal enterokinase at the residue immediately following theAsp-Lys pairing. Fusion proteins capped with this peptide may also beresistant to intracellular degradation in E. coli. A specific example ofsuch a peptide is soluble human IL-4R (1-207) Asp Tyr Lys Asp Asp AspAsp Lys.

IL-4R derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of IL-4 or other binding ligands. IL-4Rderivatives may also be obtained by cross-linking agents, such asM-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. IL-4R proteins may also be covalentlybound through reactive side groups to various insoluble substrates, suchas cyanogen bromide-activated, bisoxirane-activated,carbonyidiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, IL-4R may be used toselectively bind (for purposes of assay or purification) anti-IL-4Rantibodies or IL-4 .

The present invention also includes IL-4R with or without associatednative-pattern glycosylation. IL-4R expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or significantlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of IL-4RDNAs in bacteria such as E. coli provides non-glycosylated molecules.Functional mutant analogs of mammalian IL-4R having inactivatedN-glycosylation sites can be produced by oligonucleotide synthesis andligation or by site-specific mutagenesis techniques. These analogproteins can be produced in a homogeneous, reduced-carbohydrate form ingood yield using yeast expression systems. N-glycosylation sites ineukaryotic proteins are characterized by the amino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. In thissequence, asparagine provides a side chain amino group for covalentattachment of carbohydrate. Such a site can be eliminated bysubstituting another amino acid for Asn or for residue Z, deleting Asnor Z, or inserting a non-Z amino acid between A₁ and Z, or an amino acidother than Asn between Asn and A₁.

IL-4R derivatives may also be obtained by mutations of IL-4R or itssubunits. An IL-4R mutant, as referred to herein, is a polypeptidehomologous to IL-4R but which has an amino acid sequence different fromnative IL-4R because of a deletion, insertion or substitution. Like mostmammalian genes, mammalian IL-4 receptors are presumably encoded bymulti-exon genes. Alternative mRNA constructs which can be attributed todifferent mRNA splicing events following transcription, and which sharelarge regions of identity or similarity with the cDNAs claimed herein,are considered to be within the scope of the present invention.

Bioequivalent analogs of IL-4R proteins may be constructed by, forexample, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues can be deleted orreplaced with other amino acids to prevent formation of incorrectintramolecular disulfide bridges upon renaturation.

Other approaches to mutagenesis involve modification of adjacent dibasicamino acid residues to enhance expression in yeast systems in which KEX2protease activity is present. Generally, substitutions should be madeconservatively; i.e., the most preferred substitute amino acids arethose having physicochemical characteristics resembling those of theresidue to be replaced. Similarly, when a deletion or insertion strategyis adopted, the potential effect of the deletion or insertion onbiological activity should be considered.

Subunits of IL-4R may be constructed by deleting terminal or internalresidues or sequences.

Particularly preferred subunits include those in which the transmembraneregion and intracellular domain of IL-4R are deleted or substituted withhydrophilic residues to facilitate secretion of the receptor into thecell culture medium. The resulting protein is a soluble IL-4R moleculewhich may retain its ability to bind IL-4. Particular examples ofsoluble IL-4R include polypeptides having substantial identity tosoluble murine IL-4R (1-208), soluble human IL-4R (1-0207) and solublehuman IL-4R (1-198), all of which retain the biological activity ofsoluble human IL-4R (1-207). Chimeric polypeptides comprising fragmentsof human and murine IL-4R may also be constructed, for example, IL-4R(1-197) Pro Ser Asn Glu Asn Leu, which is comprised of the sequence ofamino acids 1-197 of human IL-4R followed by the N-terminal six aminoacids of soluble murine IL-4 R clone 18. This polypeptide has been foundto retain the biological activity of soluble IL-4R (1-207).

Mutations in nucleotide sequences constructed for expression of analogIL-4Rs must, of course, preserve the reading frame phase of the codingsequences and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structures, such as loops orhairpins, which would adversely affect translation of the receptor mRNA.Although a mutation site may be predetermined, it is not necessary thatthe nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed IL-4R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes IL-4R will beexpressed in the final product, for example, nucleotide substitutionsmay be made to enhance expression, primarily to avoid secondarystructure loops in the transcribed mRNA (see EPA 75,444A, incorporatedherein by reference), or to provide codons that are more readilytranslated by the selected host, e.g., the well-known E. coli preferencecodons for E. coli expression.

Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino aid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineenng: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated byreference herein.

Expression of Recombinant IL-4R

The present invention provides recombinant expression vectors whichinclude synthetic or cDNA-derived DNA fragments encoding mammalian IL-4R or bioequivalent analogs operably linked to suitable transcriptionalor translational regulatory elements derived from mammalian, microbial,viral or insect genes. Such regulatory elements include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation, as described in detail below. The ability to replicate in ahost, usually conferred by an origin of replication, and a selectiongene to facilitate recognition of transformants may additionally beincorporated. DNA regions are operably linked when they are functionallyrelated to each other. For example, DNA for a signal peptide (secretoryleader) is operably linked to DNA for a polypeptide if it is expressedas a precursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence it it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading frame.

DNA sequences encoding mammalian IL-4 receptors which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA; however,premature termination of transcription may be desirable, for example,where it would result in mutants having advantageous C-terminaltruncations, for example, deletion of a transmembrane region to yield asoluble receptor not bound to the cell membrane. Due to code degeneracy,there can be considerable variation in nucleotide sequences encoding thesame amino acid sequence; exemplary DNA embodiments are thosecorresponding to the nucleotide sequences shown in the Figures. Otherembodiments include sequences capable of hybridizing to the sequences ofthe Figures under moderately stringent conditions (50° C., 2×SSC) andother sequences hybridizing or degenerate to those described above,which encode biologically active IL-4 receptor polypeptides.

DNA which codes for soluble IL-4R proteins may be isolated using thecloning techniques described in the examples or may be made byconstructing cDNAs which encode only the extracellular domain of IL-4receptor (devoid of a transmembrane region) using well-known methods ofmutagenesis. Soluble forms of human IL-4 receptor are not yet known toexist and must therefore be constructed from isolated recombinant IL-4receptor cDNAs. cDNAs which encode sIL-4R may be constructed, forexample, by truncating a cDNA encoding the full length IL-4 receptor 5'of the transmembrane region, ligating synthetic oligonucleotides toregenerate truncated portions of the extracellular domain, if necessary,and providing a stop codon to terminate transcription. DNA sequencesencoding the soluble IL-4 receptor proteins can be assembled from CDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene which is capable of beingexpressed in a recombinant transcriptional unit. Such DNA sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. Genomic DNA containing the relevantsequences could also be used. Sequences of non-translated DNA may bepresent 5' or 3' from the open reading frame, where the same do notinterfere with manipulation or expression of the coding regions.

Transformed host cells are cells which have been transformed ortransfected with IL-4R vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express IL-4R, but hostcells transformed for purposes of cloning or amplifying IL-4 R DNA donot need to express IL-4R. Expressed IL-4R will be deposited in the cellmembrane or secreted into the culture supernatant, depending on theIL-4R DNA selected. Suitable host cells for expression of mammalianIL-4R include prokaryotes, yeast or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coil or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce mammalian IL-4R using RNAs derived from the DNA constructs ofthe present invention. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (Cloning Vectors: A Laboratory Manual,Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

Prokaryotic expression hosts may be used for expression of IL-4Rs thatdo not require extensive proteolytic and disulfide processing.Prokaryotic expression vectors generally comprise one or more phenotypicselectable markers, for example a gene encoding proteins conferringantibiotic resistance or supplying an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimuium, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus,although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and c1857ts thermolabile repressor. Plasmidvectors avaeiae from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Recombinant IL-4R proteins may also be expressed in yeast hosts,preferably from the Saccharomyces genus, such as S. cerevisiae. Yeast ofother genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the 2μ yeast plasmid or an autonomously replicating sequence (ARS), promoter,DNA encoding IL-4R, sequences for polyadenylation and transcriptiontermination and a selection gene. Preferably, yeast vectors will includean origin of replication and selectable marker permitting transformationof both yeast and E. coli, e.g., the ampicillin resistance gene of E.coli and S. cerevisiae trp1 gene, which provides a selection marker fora mutant strain of yeast lacking the ability to grow in tryptophan, anda promoter derived from a highly expressed yeast gene to inducetranscription of a structural sequence downstream. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described inHitzeman, EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kulan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3' end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supematants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Examples of suitable mammalian hostcell lines include the COS-7 fines of monkey kidney cells, described byGluzman (Cell 23:175, 1981), and other cell lines capable of expressingan appropriate vector including, for example, L cells, C127, 3T3,Chinese hamnsterovary (CHO), HeLa and BHK cell lines. Mammalianexpression vectors may comprise nontranscribed elements such as anorigin of replication, a suitable promoter and enhancer linked to thegene to be expressed, and other 5' or 3' flanking nontranscribedsequences, and 5' or 3' nontranslated sequences, such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the Bgl I site locatedin the viral origin of replication is included. Further, mammaliangenomic IL-4R promoter, control and/or signal sequences may be utilized,provided such control sequences are compatible with the host cellchosen. Additional details regarding the use of mammalian highexpression vectors to produce a recombinant mammalian IL-4receptor areprovided in Example 8 below. Exemplary vectors can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986).

A particularly preferred eukaryotic vector for expression of IL-4R DNAis disclosed below in Example 2. This vector, referred to as pCAV/NOT,was derived from the mammalian high expression vector pDC201 andcontains regulatory sequences from SV40, adenovirus-2, and humancytomegalovirus. pCAV/NOT containing a human IL-7 receptor insert hasbeen deposited with the American Type Culture Collection (ATCC) underdeposit accession number 68014.

Purification of IL-4 Receptors

Purified mammalian IL-4 receptors or analogs are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, and purifying IL-4receptor from the culture media or cell extracts.

For example, supematants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.For example, a suitable affinity matrix can comprise an IL-4 or lectinor antibody molecule bound to a suitable support. Alternatively, ananion exchange resin can be employed, for example, a matrix or substratehaving pendant diethylaminoethyl (DEAE) groups. The matrices can beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. Alternatively, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups arepreferred.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify an IL-4R composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian IL-4 R can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express mammalian IL-4R as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reversed-phase HPLCsteps for purification of recombinant human IL-2 on a preparative HPLCcolumn.

Human IL-4R synthesized in recombinant culture is characterized by thepresence of nonhuman cell components, including proteins, in amounts andof a character which depend upon the purification steps taken to recoverhuman IL-4R from the culture. These components ordinarily will be ofyeast, prokaryotic or non-human higher eukaryotic origin and preferablyare present in innocuous contaminant quantities, on the order of lessthan about 1 percent by weight. Further, recombinant cell cultureenables the production of IL-4R free of proteins which may be normallyassociated with IL-4R as it is found in nature in its species of origin,e.g. in cells, cell exudates or body fluids.

Administration of Soluble ILK Receptor Comsitions

The present invention provides methods of using therapeutic compositionscomprising an effective amount of soluble IL-4 receptor proteins and asuitable diluent and carrier, and methods for suppressing IL-4-dependentimmune respaces in humans comprising administering an effective amountof soluble IL-4 receptor protein.

For therapeutic use, purified soluble IL-4 receptor protein isadministered to a patient, preferably a human, for treatment in a mannerappropriate to the indication. Thus, for example, soluble IL-4 receptorprotein compositions administered to suppress immune function can begiven by bolus injection, continuous infusion, sustained release fromimplants, or other suitable technique. Typically, a soluble ILA receptortherapeutic agent will be administered in the form of a compositioncomprising purified protein in conjunction with physiologicallyacceptable carriers, excipients or diluents. Such carriers will benontoxic to recipients at the dosages and concentrations employed.Ordinarily, the preparation of such compositions entails combining theIL-4R with buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) polypeptides, proteins, aminoacids, carbohydrates including glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione and other stabilizers and excipients.Neutral buffered saline or saline mixed with nonspecific serum albuminare exemplary appropriate diluents. Preferably, product is formulated asa lyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents. Appropriate dosages can be determined in trials; generally,soluble IL-4 receptor dosages of from about 1 ng/kg/day to about 10mg/kg/day, more preferably from about 500 ng/kg/day to about 5mg/kg/day, and most preferably from about 5 ug/kg/day to about 2mg/kg/day, are appropriate for inducing a biological effect. The amountand frequency of administration will depend, of course, on such factorsas the nature and severity of the indication being treated, the desiredresponse, the condition of the patient, and so forth.

Soluble IL-4R proteins are administered for the purpose of inhibitingIL-4 dependent responses, such as suppressing immune responses in ahuman. A variety of diseases or conditions are caused by IL-4 dependentimmune responses as determined by the ability of sIL-4R to inhibit theresponse. Soluble IL-4R compositions may be used, for example, toregulate the function of B cells. Soluble IL-4R inhibits IL-4 dependentB cell proliferation and isotype specific (IgGI abd IgE) secretions.sIL-4R may therefore be used to suppress IgE antibody formation in thetreatment of IgE-induced immediate hypersensitivity reactions, such asallergic rhinitis (common hay fever), bronchial asthma, atopicdermatitis and gastrointestinal food allergy.

sIL-4R compositions may also be used to regulate the function of Tcells. Although T cell dependent functions were formerly thought to bemediated principally by IL-2, recent studies have shown that under somecircumstances T cell growth and proliferation can be mediated by growthfactors such as IL-4. Examples 20 through 23 below, for example,indicate that sIL-4R suppresses or inhibits T-cell dependent responsesto alloantigen. A variety of diseases or conditions are caused by animmune response to alloantigen, including allograft rejection andgraft-versus-host reaction. In alloantigen-induced immune responses,sIL-4R suppresses lymphoproliferation and inflammation which result uponactivation of T cells. sIL-4R has therefore been shown to be potentiallyeffective in the clinical treatment of, for example, rejection ofarografts (such as skin, kidney, heart, lung liver and pancreastransplants), and graft-versus-host reactions in patients who havereceived bone marrow transplants.

sIL-4R may also be used in clinical treatment of autoimmunedysfunctions, such as rheumatoid arthritis, diabetes, which aredependent upon the activation of T cells against antigens not recognizedas being indigenous to the host.

Because of the primary role IL-2 plays in the proliferation anddifferentiation of T cells, combination therapy using both IL-4 and IL-2may be used in the treatment of T cell dependent dysfunctions. Use inconjunction with other soluble cytokine receptors, e.g., IL-1 receptor,is also contemplated.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Binding assays for IL-4 receptor

A. Radiolabeling of IL-4. Recombinant murine and human IL-4 wereexpressed in yeast and purified to homogeneity as described by Park, etal., Proc. Natl. Acad. Sci. USA 84:5267 (1987) and Park et al., J. Exp.Med. 166-476 (1987), respectively. The purified protein was radiolabeledusing a commercially available enzymobead radioiodination reagent(BioRad). In this procedure 2.5 μg rlL-4 in 50 μIl 0.2M sodiumphosphate, pH 7.2 are combined with 50 μl enzymobead reagent, 2 MCi ofsodium iodide in 20 μl of 0.05M sodium phosphate pH 7.0 and 10 μl of2.5% b-D-glucose. After 10 min at 25° C., sodium azide (10 μl of 50 mM)and sodium metabisulfite (10 μl of 5 mg/ml) were added and incubationcontinued for 5 min. at 25° C. The reaction mixture was fractionated bygel filtration on a 2 ml bed volume of Sephadex® G-25 (Sigma)equilibrated in Roswell Park Memorial Institute (RPMI) 1640 mediumcontaining 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodiumazide and 20 mM Hepes pH 7.4 (binding medium). The final pool of ¹²⁵I-IL-4 was diluted to a working stock solution of 2×10⁻⁸ M in bindingmedium and stored for up to one month at 4° C. without detectable lossof receptor binding activity. The specific activity is routinely in therange of 1-2×10¹⁶ cprm/mmole IL-4.

B. Binding to Adherent Cells. Binding assays done with cells grown insuspension culture (i.e., CTLL and CTLL-19.4) were performed by aphthalate oil separation method (Dower et al., J. Immunol. 132:751,1984) essentially as described by Park et al., J. Biol. Chem. 261:4177,1986 and Park et al., supra. Binding assays were also done on COS cellstransfected with a mammalian expression vector containing cDNA encodingan IL-4 receptor molecule. For Scatchard analysis of binding to adherentcells, COS cells were transfected With plasmid DNA by the method ofLuthman et al., Nucl. Acids. Res. 11:1295, 1983, and McCutchan et al.,J. Natl. Cancer Inst. 41:351, 1968. Eight hours following transfection,cells were trypsinized, and reseeded in six well plates (Costar,Cambridge, Mass.) at a density of 1×10⁴ COS-IL-4 receptortransfectants/well mixed with 5×10⁵ COS control transfected cells ascarriers. Two days later monolayers were assayed for ¹²⁵ I-IL-4 bindingat 4° C. essentially by the method described by Park et al., J. Exp.Med. 166:476, 1987. Nonspecific binding of ¹²⁵ I-IL-4 was measured inthe presence of a 200-fold or greater molar excess of unlabeled IL-4 .Sodium azide (0.2%) was included in all binding assays to inhibitinternalization of ¹²⁵ I-IL-4 by cells at 37° C.

For analysis of inhibition of binding by soluble IL-4R, supernatantsfrom COS cells transfected with recombinant IL-4R constructs wereharvested three days after transfection. Serial two-fold dilutions ofconditioned media were pre-incubated with 3×10⁻¹⁰ M ¹²⁵ 1-IL-4 (having aspecific activity of about 1×10¹⁶ cpm/mmol) for one hour at 37° C. priorto the addition of 2×10⁶ CTLL cells. Incubation was continued for 30minutes at 37° C. prior to separation of free and cell-bound murine ¹²⁵IL-4.

C. Solid Phase Binding Assays. The ability of IL-4 receptor to be stablyadsorbed to nitrocellulose from detergent extracts of CTLL 19.4 cellsyet retain IL-4 binding activity provided a means of monitoringpurification. One ml aliquots of cell extracts (see Example 3), IL-4affinity column fractions (see Example 4) or other samples are placed ondry BA85121 nitrocellulose membranes (Schleicher and Schuell, Keene,N.H.) and allowed to dry. The membranes are incubated in tissue culturecishes for 30 minutes in Tris (0.05M) buffered saline (0.15M) pH 7.5containing 3% w/v BSA to block nonspecific binding sites. The membraneis then covered with 4×10⁻¹¹ M ¹²⁵ I-IL-4 in PBS+3% BSA with or withouta 200 fold molar excess of unlabeled IL-4 and incubated for 2 hr at 4°C. with shaking. At the end of this time, the membranes are washed 3times in PBS, dried and placed on Kodak X-Omat™ AR film for 18 hr at-70° C.

Example 2 Selection of CTLL cells with high IL-4 receptor expression byfluorescence activated cell sorting (FACS)

The preferred cell line for obtaining high IL-4 receptor selection isCTLL, a murine IL-2 dependent cytotoxic T cell line (ATCC TIB 214) whichtypically exhibits 2,000 to 5,000 IL-4 receptors per cell and respondsto IL-4 by short-term proliferation. To obtain higher levels of IL-4receptor expression, CTLL cells (parent cells) were subjected tomultiple rounds of fluorescence-activated cell sorting with labeledIL-4. A highly fluorescent derivative of IL-4 was derived by conjugatingrmlL-4 fluorescein hydrazide to periodate oxidized sugar moieties ofIL-4 which was produced in yeast as described by Park et al., Proc.Natl. Acad. Sci. USA 84:1669 (1987). The fluorescein-conjugated IL-4 wasprepared by combining aliquots of hyperglycosylated rmlL-4 (300 μg in300 μl of 0.1M citrate-phosphate buffer, pH 5.5) with 30 μl of 10 mMsodium m-periodiate (Sigma), freshly prepared in 0.1M citrate-phosphate,pH 5.5 and the mixture incubated at 4° C. for 30 minutes in the dark.The reaction was quenched with 30 μl of 0.1M glycerol and dialyzed for18 hours at 4° C. against 0.1M citrate-phosphate pH 5.5. Followingdialysis, a 1/10 volume of 100 mM5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminofluorescein (MolecularProbes, Eugene, Oreg.) dissolved in DMSO was added to the sample andincubated at 25° C. for 30 minutes. The IL4-fluorescein was thenexhaustively dialyzed at 4° C. against PBS, pH 7.4 and proteinconcentration determined by amino acid analysis. The final product wasstored at 4° C. following the addition of 1% (w/v) BSA and sterilefiltration.

CTLL cells (5×10⁶) were then incubated for 30 min at 37° C. in 150 μlPBS+1% BSA containing 1×10⁻⁹ M IL4-fluorescein under sterile conditions.The mixture was then chilled to 4° C., washed once in a large volume ofPBS+1% BSA and sorted using an EPICS® C. flow cytometer (CoulterInstrumerts). The cells providing the highest level fluorescence signal(top 1.0%) were collected in bulk and the population expanded in liquidcell culture and subjected to additional rounds of sorting as describedbelow. Alternatively, for single cell cloning, cells exhibiting afluorescence signal in the top 1.0% were sorted into 96 well tissueculture microtiter plates at 1 cell per well.

Progress was monitored by doing binding assays with ¹²⁵ I-L4 followingeach round of FACS selection. Unsorted CTLL cells (CTLL parent)typically exhibited 1000-2000 IL-4 receptors per cell. CTLL cells weresubjected to 19 rounds of FACS selection. The final CTLL cells selected(CTLL-19) exhibited 5×10⁵ to 1×10⁶ IL-4 receptors per cell. At thispoint the CTLL-19 population was subjected to EPICS® C-assisted singlecell cloning and individual clonal populations were expanded and testedfor ¹²⁵ I-IL4 binding. A single clone, designated CTLL-19.4, exhibited1×10⁶ IL-4 receptors per cell and was selected for purification andcloning studies. While the calculated apparent K_(a) values are similarfor the two lines, CTLL-19.4 expresses approximately 400-fold morereceptors on its surface than does the CTLL parent.

Example 3 Detergent extraction of CTLL cells

CTLL 19.4 cells were maintained in RPMI 1640 containing 10% fetal bovineserum, 50 U/ml penicillin, 50 μg/ml streptomycin and 10 ng/ml ofrecombinant human IL-2. Cells were grown to 5×10⁵ cells/ml in rollerbottles, harvested by centrifugation, washed twice in serum free DMEMand segmented at 2000×g for 10 minutes to form a packed pellet (about2×10⁸ cells/ml). To the pellet was added an equal volume of PBScontaining 1% Triton® X-100 and a cocktail of protease inhibitors (2 mMphenylmethysufonyffluoride, 10 μM pepstatin, 10 μM leupeptin, 2 mMo-phenanthroline and 2 mM EGTA). The cells were mixed with theextraction buffer by vigorous vortexing and the mixture incubated on icefor 20 minutes after which the mixture was centrifuged at 12,000×g for20 minutes at 8° C. to remove nuclei and other debris. The supernatantwas either used immediately or stored at -70° C. until use.

Example 4 IL-4 receptor purification by IL-4 affinity chromatography

In order to obtain sufficient quantities of murine IL-4R to determineits N-terminal sequence or to further characterize human IL-4R, proteinobtained from the detergent extraction of cells was further purified byaffinity chromatography. Recombinant murine or human IL-4 was coupled toAffigel®-10 (BioRad) according to the manufacturers suggestions. Forexample, to a solution of IL-4 (3.4 mg/ml in 0.4 ml of 0.1M Hepes pH7.4) was added 1.0 ml of washed Affigel®-10. The solution was rockedovernight at 4° C. and an aliquot of the supernatant tested for proteinby a BioRad protein assay per the manufacturers instructions using BSAas a standard. Greater than 95% of the protein had coupled to the gel,suggesting that the column had a final load of 1.3 mg IL-4 per ml gel.Glycine ethyl ester was added to a final concentration of 0.05M to blockany unreacted sites on the gel. The gel was washed extensively withPBS-1% Triton® followed by 0.1 Glycine-HCl, pH 3.0. A 0.8×4.0 cm columnwas prepared with IL-4-coupled Affigel® prepared as described (4.0 mlbed volume) and washed with PBS containing 1% Triton® X-100 forpurification of murine IL-4 R. Alternatively, 50 μl aliquots of 20%suspension of IL-4 coupled Affigel® were incubated with ³⁵S-cysteine/methionine-labeled cell extracts for small-scale affinitypurifications and gel electrophoresis.

Aliquots (25 ml) of detergent extracted IL-4 receptor bearing CTLL 19.4cells were slowly applied to the murine IL-4 affinity column at 4° C.(flow rate of 3.0 ml/hr). The column was then washed sequentially withPBS containing 1% Triton® X-100, RIPA buffer (0.05M Tris, 0.15M NaCl, 1%NP-40, 1% deoxycholate and 0.1% SDS), PBS containing 0.1% Triton® X-100and 10 mM ATP, and PBS with 1% Triton® X-100 to remove all contaminatingmaterial except the m/L-4R. The column was then eluted with pH 3.0glycine HCl buffer containing 0.1% Triton® X-100 to remove the IL-4R andwashed subsequently with PBS containing 0.1% Triton® X-100. Onemilliliter fractions were collected for the elution and 2 ml fractionscollected during the wash. Immediately following elution, samples wereneutralized with 80 μl of 1M Hepes, pH 7.4. The presence of receptor inthe fractions was detected by the solid phase binding assay as describedabove, using ¹²⁵ I-labeled IL-4 . Aliquots were removed from eachfraction for analysis by SDS-PAGE and the remainder frozen at -70° C.until use. For SDS-PAGE, 40 μl of each column fraction was added to 40μl of 2×SDS sample buffer (0.125M Tris HCl pH 6.8, 4% SDS, 20% glycerol,10% 2-mercaptoethanol). The samples were placed in a boiling water bathfor 3 minutes and 80 μl aliquots applied to sample wells of a 10%polyacrylamide gel which was set up and run according to the method ofLaemmli (Nature 227:680, 1970). Following electrophoresis, gels weresilver stained as previously described by Urdal et al. (Proc. Natl.Acad. Sci. USA 81:6481, 1984).

Purification by the foregoing process permitted identification by silverstaining of polyacrylamide gels of two mIL4R protein bands averaging45-55 kDa and 30-40 kDa that were present in fractions exhibiting IL-4binding activity. Experiments in which the cell surface proteins ofCTLL-19.4 cells were radiolabeled and ¹²⁵ -labeled receptor was purifiedby affinity chromatography suggested that these two proteins wereexpressed on the cell surface. The ratio of the lower to highermolecular weight bands increased upon storage of fractions at 4° C.,suggesting a precursor product relationship, possibly due to slowproteolytic degradation. The mlL-4 receptor protein purified by theforegoing process remains capable of binding IL-4 , both in solution andwhen adsorbed to nitrocellulose.

Example 5 Sequencing of IL-4 receptor protein

CTLL 19.4 mIL-4 receptor containing fractions from the mIL-4 affinitycolumn purification were prepared for amino terminal protein sequenceanalysis by fractionating on an SDS-PAGE gel and then transferred to aPVDF membrane. Prior to running the protein fractions on polyacrylamidegels, it was first necessary to remove residual detergent from theaffinity purification process. Fractions containing proteins bound tothe mlL-4 affinity column from three preparations were thawed andconcentrated individually in a speed vac under vacuum to a final volumeof 1 ml. The concentrated fractions were then adjusted to pH 2 by theaddition of 50% (vN) TFA and injected onto a Brownlees RP-300reversed-phase HPLC column (2.1×30 mm) equilibrated with 0.1% (v/v) TFAin H₂ O at a flow rate of 200 μl/min running on a Hewlett Packard Model1090M HPLC. The column was washed with 0.1% TFA in H₂ O for 20 minutespost injection. The HPLC column containing the bound protein was thendeveloped with a gradient as follows:

    ______________________________________    Time      % Acetonitrile in 0.1% TFA    ______________________________________     0        0     5        30    15        30    25        70    30        70    35        100    40        0    ______________________________________

1 ml fractions were collected every five minutes and analyzed for thepresence of protein by SDS PAGE followed by silver staining.

Each fraction from the HPLC run was evaporated to dryness in a speed vacand then resuspended in Laemmli reducing sample buffer, prepared asdescribed by Laemmli, U.K. Nature 227.680, 1970. Samples were applied toa 5-20% gradient Laemmli SDS gel and run at 45 mA until the dye finreached the bottom of the gel. The gel was then transferred to PVDFpaper and stained as described by Matsudaira, J. Biol. Chem. 262:10035,1987. Staining bands were clearly identified in fractions from each ofthe three preparations at approximately 30,000 to 40,000 M_(r).

The bands from the previous PVDF blotting were excised and subjected toautomated Edman degradation on an Applied Biosystems Model 477A ProteinSequencer essentially as described by March et al. (Nature 315:641,1985), except that PTH amino acids were automatically injected andanalyzed on line with an Applied Biosystems Model 120A HPLC using agradient and detection system supplied by the manufacturer. Thefollowing amino terminal sequence was determined from the results ofsequencing: NH₂-Ile-Lys-Val-Leu-Gly-Gk-Pro-Thr-(Cys/Asn)-Phe-Ser-Asp-Tyr-Ile. Position9 was assigned as a cysteine or glycosylated asparagine owing to thelack of an observable PTH-amino acid signal in the cycle. The bands fromthe second preparation used for amino terminal sequencing were treatedwith CNBr using the in situ technique described by March et al. (Nature315: 641, 1985) to cleave the protein after internal methionineresidues. Sequencing of the resulting clleavage products yielded thefollowing data, indicating that the CNBr cleaved the protein after twointernal methionine residues:

    ______________________________________    Cycle             Residues Observed    ______________________________________    1                 Val, Ser    2                 Gly, Leu    3                 Ile, Val    4                 Tyr, Ser    5                 Arg, Tyr    6                 Glu, Thr    7                 Asp, Ala    8                 Asn, Leu    9                 Pro, Val    10                Ala    11                Glu, Val    12                Phe, Gly    13                Ile, Asn    14                Val, Gln    15                Tyr, Ile    16                Lys, Asn    17                Val, Thr    18                Thr, Gly    ______________________________________

When compared with the protein sequences derived from clones 16 and 18(see FIG. 2), the sequences matched as follows: ##STR1## Identicalmatches were found for all positions of sequence 1 except Asn(2) andsequence 2, except Arg at positions 8, 10, and 12, Ser at position 13,and Leu at position 16. The above sequences correspond to amino acidresidues 137-154 and 169-187 of FIG. 2A.

In addition, the amino terminal sequence matched a sequence derived fromthe clone with position 9 being defined as a Cys.

The above data support the conclusion that clones 16 and 18 are derivedfrom the message for the IL-4 receptor.

Example 6 Synthesis of hybrid-subtracted cDNA probe

In order to screen a library for clones encoding a murine IL-4 receptor,a highly enriched IL-4 receptor CDNA probe was obtained using asubtractive hybridization strategy. Polyadenylated (polyA+) mRNA wasisolated from two similar cell lines, the parent cell line CTLL (whichexpresses approximately 2,000 receptors per cell) and the sorted cellline CTLL 19.4 (which expresses 1×10⁶ receptors per cell). The mRNAcontent of these two cell lines is expected to be identical except forthe relative level of IL-4 receptor mRNA. A radiolabeled single-strandedcDNA preparation was then made from the mRNA of the sorted cell lineCTLL 19.4 by reverse transcription of polyadenylated mRNA from CTLL 19.4cells by a procedure similar to that described by Maniatis et al.,Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory,New York, 1982). Briefly, polyA+mRNA was purified as described by Marchet al. (Nature 315:641-647, 1985) and copied into cDNA by reversetranscriptase using oligo dT as a primer. To obtain a high level of ³²P-labeling of the cDNA,100 μCi of ³² P-dCTP (s.a.=3000 Ci/mmol was usedin a 50 μl reaction with non-radioactive dCTP at 10 μM. After reversetranscription at 42° C. for 2 hours, EDTA was added to 20 mM and the RNAwas hydrolyzed by adding NaOH to 0.2M and incubating the CDNA mixture at68° C. for 20 minutes. The single-stranded cDNA was extracted with aphenouchloroform (50/50) mixture previously equilibrated with 10 mMTris-CI, 1 mM EDTA. The aqueous phase was removed to a clean tube andmade alkaline again by the addition of NaOH to 0.5M. The CDNA was thensize-fractionated by chromatography on a 6 ml Sephadex® G50 column in 30mM NaOH and 1 mM EDTA to remove small molecular weight contaminants.

The resulting size-fractionated cDNA generated from the sorted CTLL 19.4cells was then hybridized with an excess of mRNA from the unsortedparental CTLL cells by ethanol-precipitating the CDNA from CTL 19-4cells with 30 μg of polyA+mRNA isolated from unsorted CTLL cells,resuspending in 16 μl of 0.25M NaPO₄, pH 6.8, 0.2% SDS, 2 mM EDTA andincubating for 20 hours at 68° C. The cDNAs from the sorted CTLL 19.4cells that are complementary to mRNAs from the unsorted CTLL cells formdouble stranded cDNA/mRNA hybrids, which can then be separated from thesingle stranded cDNA based on their different binding affinities onhydroxyapatite. The mixture was diluted with 30 volumes of 0.02M NaPO₄,pH 6.8, bound to hydroxyapatite at room temperature, and single-strandedcDNA was then eluted from the resin with 0.12M NaPO₄, pH 6.8, at 60° C.,as described by Sims et al., Nature 312:541, 1984. Phosphate buffer wasthen removed by centrifugation over 2 ml Sephadex® G50 spin columns inwater. This hybrid subtraction procedure removes a majority of commonsequences between CTLL 19.4 and unsorted CTLL cells, and leaves asingle-stranded CDNA pool enriched for radiolabeled IL-4 receptor CDNAwhich can be used to probe a cDNA library (as described below).

Example 7 Synthesis of CDNA library and plaque screening

A cDNA library was constructed from polyadenylated mRNA isolated fromCTLL 19.4 cells using standard techniques (Gubler, et al-, Gene 25:263,1983; Ausubel et al., eds., Current Protocols in Molecular Biology, Vol.1, 1987). After reverse transcription using oligo dT as primer, thesingle-stranded CDNA was itndered douse-stranded with DNA polymerase I,blunt-ended with T4 DNA polymerase, methylated with EcoR I methylase toprotect EcoR I cleavage sites within the cDNA, and ligated to EcoR Ilinkers. The resulting constructs were digested with EcoR I to removeall but one copy of the linkers at each end of the cDNA, and ligated toan equimolar concentration of EcoR I cut and dephosphorylated λAP® armsand the resulting ligation mix was packaged in vitro (Gigapack®)according to the manufacturer's instructions. Other suitable methods andreagents for generating cDNA libraries in λ phage vectors are describedby Huynh et al., DNA Cloning Techniques: A Practical Approach, IRLPress, Oxford 1984), Meissner et al., Proc. Natl. Acad. Sci. USA 84:4171(1987), and Ausubel et al., supra. λZAP® is a phage λ cloning vectorsimilar to λgt11 (U.S. Pat. No. 4,788,135) containing plasmid sequencesfrom pUC19 (Norrander et al., Gene 26:101, 1987), a polylinker sitelocated in a lacZ gene fragment, and an f1 phage origin of replicationpermitting recovery of ssDNA when host bacteria are superinfected withf1 helper phage. DNA is excised in the form of a plasmid comprising theforegoing elements, designated Bluescript®. Gigapack® is a sonicated E.coli extract used to package λ phage DNA. λZAP®, Bluescript®, andGigapack® are registered trademarks of Stratagene, San Diego, Calif.,USA.

The radiolabeled hybrid-subtracted cDNA from Example 6 was then used asa probe to screen the CDNA library. The amplified library was plated onBB4 cells at a density of 25,000 plaques on each of 20 150 mm plates andincubated overnight at 37° C. All manipulations of λZAP® and excision ofthe Bluescript® plasmid were as described by Short et al., (Nucl. AcidsRes. 16.7583, 1988) and Stratagene product literature. Duplicate plaquelift filters were incubated with hybrid-subtracted cDNA probes fromExample 6 in hybridization buffer containing 50% formamide, 5×SSC,5×Denhardt's reagent and 10% dextran sulfate at 42° C. for 48 hours asdescribed by Wahl et al., Proc. Natl. Acad. Sci. USA76,3683, 1979.Filters were then washed at 68° C. in 0.2×SSC. Sixteen positive plaqueswere purified for further analysis.

Bluescript® plasmids containing the cDNA inserts were excised from thephage as described by the manufacturer and transformed into E coli.Plasmid DNA was isolated from individual colonies, digested with EcoR Ito release the CDNA inserts and electrophoresed on standard 1% agarosegels. Four duplicate gels were blotted onto nylon filters to produceidentical Southern blots for analysis with various probes which were (1)radiolabeled cDNA from unsorted CTLL cells, (2) radiolabeled cDNA fromCTLL 19.4 sorted cells, (3) hybrid subtracted cDNA from CTLL 19.4 sortedcells, and (4) hybrid subtracted cDNA from CTLL 19.4 sorted cells aftera second round of hybridization to poly A⁺ mRNA from an IL-4 receptornegative mouse cell line (LBRM 33 IASB6). These probes were increasinglyenriched for cDNA copies of MRNA specific for the sorted cell line CTLL19.4. Of the 16 positive plaques isolated from the library, four clones(11A, 14, 16 and 18) showed a parallel increase in signal strength withenrichment of the probe.

Restriction mapping (shown in FIG. 1) and DNA sequencing of the isolatedCTLL clones indicated the existence of at least two distinct mRNApopulations. Both mRNA types have homologous open reading frames overmost of the coding region yet diverge at the 3' end, thus encodinghomologous proteins with different COOH-terminal sequences. DNA sequencefrom inside the open reading frames of both clones code for proteinsequence that is identical to protein sequence derived from sequencingof the purified IL-4 receptor described in more detail in Example 5.Clone 16 and clone 18 were used as the prototypes for these two distinctmessage types. Clone 16 contains an open reading frame that encodes a258-amino acid polypeptide which includes amino acids -25 to 233 of FIG.2A. Clone 18 encodes a 230-amino acid soluble receptor protein, theN-terminal 224 amino acids of which are identical to the N-terminus ofclone 16 but diverge 9 amino acids upstream of the putativetransmembrane region beginning with nucleotide number 598. Thisinsertion adds the 3' nucleotide sequence CCAAGTAATGAAAATCTG whichencodes the C-terminal 6 amino acids, Pro-Ser-Asn-Glu-Asn-Leu, followedby a termination codon TGA. Both clones were expressed in a mammalianexpression system, as described in Example 8.

Example 8 Expression of IL-4R in mammalian cells

A. Expression in COS-7 Cells. A eukaryotic expression vector pCAV/NOT,shown in FIG. 3, was derived from the mammalian high expression vectorpDC201, described by Sims et al., Science 241:585, 1988). pDC201 is aderivative of pMLSV, previously described by Cosman et al., Nature312:768, 1984. pCAV/NOT is designed to express cDNA sequences insertedat its multiple cloning site (MCS) when transfected into mammalian cellsand includes the following components: SV40 (hatched box) contains SV40sequences from coordinates 5171-270 including the origin of replication,enhancer sequences and early and late promoters. The fragment isoriented so that the direction of transcription from the early promoteris as shown by the arrow. CMV contains the promoter and enhancer regionsfrom human cytomegalovirus (nucleotides -671 to +7 from the sequencepublished by Boshart et al., Cell 41:521-530, 1985). The tripartiteleader (stippled box) contains the first exon and part of the intronbetween the first and second exons of the adenovirus-2 tripartiteleader, the second exon and part of the third exon of the tripartiteleader and a multiple cloning site (MCS) containing sites for Xho I, Kpn1, Sma I, Not I and Bgl II. pA (hatched box) contains SV40 sequencesfrom 4127-4100 and 2770-2533 that include the polyadenylation andtermination signals for early transcription. Clockwise from pA areadenovirus-2 sequences 10532-11156 containing the VAI and VAII genes(designated by a black bar), followed by pBR322 sequences (solid line)from 4363-2486 and 1094-375 containing the ampicillin resistance geneand origin of replication. The resulting expression vector wasdesignated pCAV/NOT.

Inserts in clone 16 and clone 18 were both released from Bluescript®plasmid by digestion with Asp 718 and Not I. The 3.5 kb insert fromclone 16 was then ligated directly into the expression vector pCAVINOTalso cut at the Asp 718 and Not I sites in the polylinker region. Theinsert from clone 18 was blunt-ended with T4 polymerase followed byligation into the vector PCAV/NOT cut with Sma I and dephosphorylated.

Plasmid DNA from both IL-4 receptor expression plasmids were used totransfect a sub-confluent layer of monkey COS-7 cells using DEAE-dextranfollowed by chloroquine treatment, as described by Luthman et al. (Nucl.Acids Res. 11:129511983) and McCutchan et al. (J. Natl. Cancer lnst.41:351, 1968). The cells were then grown in culture for three days topermit transient expression of the inserted sequences. After three days,cell culture supematants and the cell monolayers were assayed (asdescribed in Example 1) and IL-4 binding was confirmed.

B. Expression in CHO Cells. IL-4 R was also expressed in the mammalianCHO cell line by first ligating an Asp718/Notl restriction fragment ofclone 18 into the pCAV/NOT vector as described in Example 8. The pCAVOTvector containing the insert from clone 18 was then co-transfected usinga standard calcium phosphate method into CHO cells with thedihydrofolate reductase (DHFR) cDNA selectable marker under the controlof the SV40 early promoter. The DHFR sequence enables methotrexateselection for mammalian cells harboring the plasmid. DHFR sequenceamplification events in such cells were selected using elevatedmethotrexate concentrations. In this way, the contiguous DNA sequencesare also amplified and thus enhanced expression is achieved. Mass cellcultures of the transfectants secreted active soluble IL-4R atapproximately 100 ng/ml.

C. Expression in HeLa Cells. IL-4R was expressed in the human HeLa-EBNAcell line 653-6, which constitutively expresses EBV nuclear antigen-1driven from the CMV immediate-early enhancer/promoter. The expressionvector used was pHAV-EO-NEO, described by Dower et al., J. Immunol.142:4314, 1989), a derivative of pDC201, which contains the EBV originof replication and allows high level expression in the 653-6 cell line.pHAV-EO-NEO is derived from pDC201 by replacing the adenovirus majorlate promoter with synthetic sequences from HIV-1 extending from -148 to+78 relative to the cap site of the viral mRNA, and including the HIV-1tat gene under the control of the SV-40 early promoter. It also containsa Bgl II-Sma I fragment containing the neomycin resistance gene ofpSV2NEO (Southern & Berg, J. MoL Appl. Genet. 1:332, 1982) inserted intothe Bgl II and Hpa I sites and subeloning downstream of the Sal Icloning site. The resulting vector permits selection of transfectedcells for neomycin resistance.

A 760 bp IL-4 R fragment of clone C-18 from the CTLL 19.4 library wasreleased from the Bluescript® plasmid of the λZAP® cloning system(Stratagene, San Diego, Calif., USA) by digesting with EcoN I and Sst Irestriction enzymes. This fragment of clone C-18 corresponds to thenucleotide sequence set forth in FIG. 2, with the addition of a 5'terminal nucleotide sequence of TGCAGGCACCTT TGTGTCCCCA, a TGA stopcodon which follows nucleotide 615 of FIG. 2A, and a 3' terminalnucleotide sequence of CTGAGTGACCTTGGGGGCTGCGGTGGTGAGGAGAGCT. Thisfragment was then blunt-ended using T4 polymerase and subcloned into theSal I site of pHAV-EO-NEO. The resulting plasmid was then transfectedinto the 653-6 cell line by a modified polybrene transfection method asdescribed by Dower et al. (J. Immunol. 142-4314, 1989) or byelectroporation with the exception that the cells were trypsinized at 2days post-transfection and split at a ratio of 1:8 into media containingG418 (Gbco Co.) at a concentration of 1 mg/mlr. Culture media werechanged twice weekly until neomycin-resistant colonies were established.Colonies were then either picked individually using cloning rings, orpooled together, to generate mass cultures. These cell lines weremaintained under drug selection at a G418 concentration of 250 ug/ml.

In an effort to select cell colonies expressing high levels of solubleIL-4 receptor, a membrane filter assay was set up as follows. Highexpressing cell clones were isolated by seeding 450 HeLa IL-4Ltransfectant cells in a 20 cm plate and allowing the cells to grow for10 days. Cell monolayers were then washed with a Tris-buffered saline(TBS) solution and overlayed with a nitrocellulose membrane. The overlaytechnique is essentially that of McCracken and Brown, Biotechniques2:82, 1984, except that the nitrocellulose was overlayed with smallglass beads to ensure that the membrane was kept flat. Cells wereincubated for an additional 24 hours to allow secretion and adsorptionof soluble IL-4 receptor to the nitrocellulose membrane. Finally themembrane was removed, washed gently in TBS containing 1% bovine serumalbumin (BSA) for 30 minutes at room temperature, then incubated in TBSwith 3% BSA containing ¹²⁵ I-IL-4(4×10⁻¹¹ M, specific activity -1×10¹⁶cpm/mmol) for two hours at 4° C. Membranes were then washed 3 times withPBS, dried and exposed on Kodak X-mat™ film overnight at -70° C.

The developed film showed spots aligned with cells growing on the platesin culture. Cell colonies aligned with the darkest spots on the film(indicating the highest level of IL-4 receptor production by cells) wereharvested, and grown up in culture. When the individual clones reachedconfluency, supernatants were tested for the presence of soluble IL-4receptor in a binding inhibition assay as follows. Inhibition assayswere performed by first incubating various concentrations of unlabeledIL-4 or soluble IL-4 receptor with 50 μl of ¹²⁵ IL-4 (1.65×10⁻¹⁰ M) forthirty minutes at 37° C. in binding medium (RPMI with 2.5% BSA, 0.20%sodium azide, 0.2% M Hepes, pH 7.4). Subsequently 2×10⁶ CTLL-2 cellswere added in 50 ul of binding medium and the incubation continued foran additional thirty minutes. Free and cell-bound ¹²⁵ I-IL-4 were thenseparated by the pthalate oil separation method (Dower, S. K. et al., J.Immunol 132:751, 1984). Percent specific inhibition was calculated usingincubation of ¹²⁵ I-IL-4 with excess unlabeled IL-4 (4×10⁻⁹ M) as apositive control and 50 ul of binding medium as a negative control. Thebinding data were calculated and graphed using RS/1 (BBN SoftwareProducts, Cambridge, Mass.) as previously described (Dower, S. K. etal., J. Exp. Med 162:501, 1985).

Initial cultures of cells produced ˜100-600 ng/ml of soluble IL-4Rprotein, and several cell clones isolated with the membrane-trappingtechnique produced as much as 2-3 ug/ml of IL-4R protein. These celllines are currently maintained under dnug selection in G418 at aconcentration of 250 ug/ml. The establishment of a stable cell clone,HeLa E3C3, producing soluble IL-4 R enabled us to begin scaling upproduction and purification of the soluble recombinant IL-4 receptor.For soluble IL-4 receptor production, the HeLa E3C3 cells were seeded inexpanded surface area roller bottles (1:20 split ratio), and were grownfor four days with 250 ml of modified Dulbecco's Eagles medium 5% fetalbovine serum and 1% penicillin, streptomycin and glutamine. Rollerbottles were then switched to serum free media (300 ml/roller bottle)for three days. Soluble IL-4 receptor protein was purified from HeLaE3C3 culture supernatants by affinity chromatography on IL-4 linked toAffigel-10. Recombinant mutine IL-4 was coupled to Affigel®-10 (BioRad)according to the manufacturer's suggestions. Briefly, 1.0 ml of washedAffigel®-10 was added to a solution of IL-4 (3.4 mg/ml in 0.4 ml of 0.1MHepes pH 7.4). The solution was rocked overnight at 4° C. and an aliquotof the supernatant tested for protein by a BioRad protein assay per themanufacturer's instructions using BSA as a standard. Greater than 95% ofthe protein had coupled to the gel, suggesting that the column had afinal load of 1.3 mg IL-4 per ml gel. Glycine ethyl ester was added to afinal concentration of 0.05M to block any unreacted sites on the gel.The gel was washed extensively with PBS-1% Triton® followed by 0.1Glycine-HCl, pH 3.0. A 0.8×4.0 cm column was prepared with IL-4 coupledAffigel® prepared as described (4.0 ml bed volume) and washed with PBScontaining 1% Triton® X-100 for purification of murine IL-4R.Alternatively, 50 μl aliquots of 20% suspension of IL4-coupled Affigel®were incubated with ³⁵ S-cysteine/methionine-labeled cell extracts forsmall-scale affinity purifications and gel electrophoresis.

Aliquots (25 ml) of HeLa E3C3 culture supernatants (containing solubleIL-4 receptor) were slowly applied to the murine IL-4 affinity column at4° C. (flow rate of 3.0 ml/hr). The column was then washed sequentiallywith PBS to remove all contaminating material except the bound mlL-4R.The column was then eluted with 0.01M acetic acid, 0.15M sodiumchloride, pH 3.0 to remove the IL-4R and washed subsequently with PBS.One ml fractions were collected for the elution and 2 ml fractionscollected during the wash. Immediately following elution, samples wereneutralized with 80 ul of 1M Hepes, pH 7.4. The presence of receptor inthe fractions was detected by the inhibition binding assay describedabove.

From 100 ml of HeLa E3C3 culture supernatant approximately 600 ug ofsoluble IL-4 receptor protein was purified on a 4.0 ml affinity column.Purified receptor consisted of three major bands ranging from 30-39,000daltons on SDS-PAGE. Heterogeneity in size of this preparation is due tovariability in protein glycosylation, as treatment of the protein withN-glycanase to remove N-linked carbohydrates reduces the size of theprotein to ˜25,000 daltons on SDS-PAGE. In addition, amino acidsequencing confirmed that the bands have the same N-terminal sequence.Purity and protein concentrations were also confirmed by amino acidanalysis.

Example 9 Expression of IL-4R in yeast cells

For expression of mIL-4R, a yeast expression vector derived from pIXY 20was constructed as follows. plXY120 is identical to pYxHuGM (ATCC53157), except that it contains no cDNA insert and includes apolylinker/multiple cloning site with an Nco I site. This vectorincludes DNA sequences from the following sources: (1) a large Sph I(nucleotide 562) to EcoR I (nucleotide 4361) fragment excised fromplasmid pBR322 (ATCC 37017), including the origin of replication and theampicillin resistance marker for selection in E. coli (2) S. cereviiaeDNA including the TRP-1 marker, 2 μ origin of replication, ADH2promoter; and (3) DNA encoding an 85 amino acid signal peptide derivedfrom the gene encoding the secreted peptide α-factor (See Kurian et al.,U.S. Pat. No. 4,546,082). An Asp 718 restriction site was irked atposition 237 in the α-factor signal peptide to facilitate fusion toheterologous genes. This was achieved by changing the thymidine residueat nucleotide 241 to a cytosine residue by oligonucleotide-directed invitro mutagenesis as described by Craik, BioTechniques, January 1985,pp. 12-19. A synthetic oligonucleotide containing multiple cloning sitesand having the following sequence was inserted from the Asp718 site atamino acid 79 near the 3' end of the α-factor signal peptide to a Spelsite in the 2μ sequence: ##STR2## pBC120 also varies from pYaHuGM by thepresence of a 514 bp DNA fragment derived from the single-stranded phagef1 containing the origin of replication and intergenic region, which hasbeen inserted at the Nru I site in the pBR322 sequence. The presence ofan f1 origin of replication permits generation of single-stranded DNAcopies of the vector when transformed into appropriate strains of E.coli and superinfected with bacteriophage f1, which facilitates DNAsequencing of the vector and provides a basis for in vitro mutagenesis.To insert a cDNA, pIXY1 20 is digested with Asp 718 which cleaves nearthe 3' end of the a-factor leader peptide (nucleotide 237) and, forexample, BamH I which cleaves in the polylinker. The large vectorfragment is then purified and ligated to a DNA fragment encoding theprotein to be expressed.

To create a secretion vector for expressing mIL-4R, a cDNA fragmentencoding mlL-4R was excised from the Bluescript® plasmid of Example 8 bydigestion with Ppum I and BgI II to release an 831 bp fragment from thePpum I site (see FIGURE) to an Bgl II site located 3' to the openreading frame containing the mIL-4R sequence minus the first two 5'codons encoding lie and Lys. plXY120 was digested with Asp 718 near the3' end of the α-factor leader and BamH I. The vector fragment wasligated to the Ppum IlBgl II hlL-4R cDNA fragment and the followingfragment created by annealing a pair of synthetic oligonucleotides torecreate the last 6 amino acids of the α-factor leader and the first twoamino acids of mature mIL-4R. ##STR3##

The oligonucleotide also included a change from the nucleotide sequenceTGG ATA to CTA GAT which introdces a Xba I restriction site, withoutaltering the encoded amino acid sequence.

The foregoing expression vector was then purified and employed totransform a diploid yeast strain of S. cerevisiae (XV2181) by standardtechniques, such as those disclosed in EPA 165,654, selecting fortryptophan prototrophs. The resulting transformants were cultured forexpression of a secreted mIL-4R protein. Cultures to be assayed forbiological activity were grown in 20-50 ml of YPD medium (1% yeastextract, 2% peptone, 1% glucose) at 37° C. to a cell density of 1-5×10⁸cells/ml. To separate cells from medium, cells were removed bycentrifugation and the medium filtered through a 0.45 μ celluloseacetate filter prior to assay. Supernatants produced by the transformedyeast strain, or crude extracts prepared from disrupted yeast cellstransformed the plasmid, were assayed to verify expression of abiologically active protein.

Example 10 Isolation of full-length and truncated forms of murine IL-4receptor cDNAs from unsorted 789 cells

Polyadenylated RNA was isolated from 789 cells, an antigen-dependenthelper T cell clone derived from C57BL/6 mice, and used to construct acDNA library in λZAP (Stratagene, San Diego), as described in Example 7.The λZAP library was amplified once and a total of 300,000 plaques werescreened as described in Example 7, with the exception that the probewas a randomly primed ³² P-labeled 700 bp EcoRI fragment isolated fromCTLL 19.4 clone 16. Thirteen clones were isolated and characterized byrestriction analysis.

Nucleic acid sequence analysis of clone 7B9-2 revealed that it containsa polyadenylated tail, a putative polyadenylation signal, and an openreading frame of 810 amino acids (shown in FIG. 2), the first 258 ofwhich are identical to those encoded by CTLL 19.4 clone 16, includingthe 25 amino acid putative signal peptide sequence. The 7B9-2 cDNA wassubcloned into the eukaryotic expression vector, pCAV/NOT, and theresulting plasmid was transfected into COS-7 cells as described inExample 8. COS-7 transfectants were analyzed as set forth in Example 12.

A second cDNA form, similar to clone 18 in the CTLL 19.4 library, wasisolated from the 7B9 library and subjected to sequence analysis. ThiscDNA, clone 7B9-4, is 376 bp shorter than clone 7B9-2 at the 5' end, andlacks the first 47 amino acids encoded by 7B9-2, but encodes theremaining N-terminal amino acids 23-199 (in FIG. 2). At position 200,clone 7B9-4 (like clone 18 from CTLL 19.4) has a 114 bp insert whichchanges the amino acid sequence to Pro Ser Asn Glu Asn Leu followed by atermination codon. The 114 bp inserts, found in both clone 7B9-4 andCTLL 19.4 clone 18 are identical in nucleic acid sequence. The fact thatthis cDNA form, which produces a secreted form of the IL-4 receptor whenexpressed in COS-7 cells, was isolated from these two different celllines indicates that it is neither a cloning artifact nor a mutant formpeculiar to the sorted CTLL cells.

Example 11 Isolation of human IL-4 receptor cDNAs from PBL and T22libraries by cross-species hybridization

Polyadenylated RNA was isolated from pooled human peripheral bloodlymphocytes (PBL) that were obtained by standard Ficoll purification andwere cultured in IL-2 for six days followed by stimulation with PMA andCon-A for eight hours. An oligo dT primed CDNA library was constructedin λgt10 using techniques described in example 7. A probe was producedby synthesizing an unlabeled RNA transcript of the 7B9-4 CDNA insertusing T7 RNA polymerase, followed by ³² P-labeled CDNA synthesis withreverse transcriptase using random primers (Boehringer-Mannheim). Thismurine single-stranded cDNA probe was used to screen 50,000 plaques fromthe human cDNA library in 50% formamide/0.4 M NaCl at 42° C., followedby washing in 2×SSC at 55° C. Three positive plaques were purified, andthe EcoR I inserts subcloned into the Bluescript® plasmid vector.Nucleic acid sequencing of a portion of clone PBL-1, a 3.4 kb cDNA,indicated the clone was approximately 67% homologous to thecorresponding sequence of the murine IL-4 receptor. However, an insertof 68 bp, containing a termination codon and bearing no homology to themouse IL-4 receptor clones, was found 45 amino acids downstream of thepredicted N-terminus of the mature protein, suggesting that clone PBL-1encodes a non-functional truncated form of the receptor. Nine additionalhuman PBL clones were obtained by screening the same library (understringent conditions) with a ³² P-labeled random-primed probe made fromthe clone PBL-1 (the 3.4 kb EcoR I cDNA insert). Two of these clones,PBL-11 and PBL-5, span the 5' region that contains the 68 bp insert inPBL-1, but lack the 68 bp insert and do not extend fully 3', asevidenced by their size, thus precluding functional analysis bymammalian expression. In order to obtain a construct expressible inCOS-7 cells, the 5' Not I-Hinc II fragment of clones PBL-11 and PBL-5were separately ligated to the 3' Hinc Il-BamH I end of clone PBL-1, andsubcloned into the pCAV/NOT expression vector cut with Not I and Bgl IIdescribed in Example 8. These chimeric human IL-4 R cDNAs containingPBL-11/PBL-1 and PBL5/PBL-1 DNA sequences have been termed clones A5 andB4, respectively, as further described in Example 12. These constructswere transfected into COS-7 cells, and assayed for IL-4 binding in aplate binding assay substantially as described in Sims et al. (Science241:585, 1988). Both composite constructs encoded protein whichexhibited IL-4 binding activity. The nucleotide sequence and predictedamino acid sequence of the composite A5 construct correspond to thesequence information set forth in FIGS. 4A-4C, with the exception that aGTC codon encodes the amino acid Val at position 50, instead of lie. Noother clones that were sequenced contained this change. The consensuscodon from clones PBL-1, PBL-5 and T22-8, however, is ATC and encodesIle⁵⁰, as set forth in FIG. 4A. The nucleotide and predicted amino acidsequence of the composite B4 construct also shows that the 25 amino acidleader sequence of PBL-11 is replaced with the sequenceMet-GIn-Lys-AspAla-Arg-Arg-Glu-Gly-Asn.

Constructs expressing a soluble form of the human IL-4 receptor weremade by excising a 5'-terminal 0.8 kb Sma I-Dra IlIl fragment from PBL-5and the corresponding 0.8 kb Asp718-Dra III fragment from PBL-11, ofwhich the Dra III overhangs were blunt-ended with T4 polymerase. ThePBL-5 and PBL-11 fragments were separately subcloned into CAVINOT cutwith Sma I or Asp 718 plus Sma I, respectively; these are called solublehlL-4R-5 and soluble hlL-4R-11, respectively.

A second library made from a CD4+/CD8- human T cell clone, T22, (Acreset al., J. Immunol. 138:2132, 1987) was screened (using duplicatefilters) with two different probes synthesized as described above. Thefirst probe was obtained from a 220 bp Pvu II fragment from the 5' endof clone PBL-1 and the second probe was obtained from a 300 bp Pvu II-EcoR I fragment from the 3' end of clone PBL-1. Five additional cDNAclones were identified using these two probes. Two of these clones spanthe 5' region containing the 68 bp insert, but neither contain theinsert. The third of these clones, T22-8, was approximately 3.6 kb insize and contained an open reading frame of 825 amino acids, including a25 amino acid leader sequence, a 207 amino acid-mature external domain,a 24 amino acid transmembrane region and a 569 amino acid cytoplasmicdomain. The sequence of clone T22-8 is set forth in FIGS. 4A-4C. FIGS.5A-5B compare the predicted human IL-4 R amino acid sequence with thepredicted murine IL-4R sequence and show approximately 53% sequenceidentity between the two proteins.

Example 12 Analysis and purification of IL-4 receptor in COStransfectants

Equilibrium binding studies were conducted for COS cells transfectedwith murine IL-4 receptor clones 16 and 18 from the CTLL 19.4 library.In all cases analysis of the data in the Scatchard coordinate system(Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949) yielded a straightline, indicating a single class of high-affinity receptors for murineIL-4 . For COS pCAV-16 cells the calculated apparent K_(a) was 3.6×10⁹M⁻¹ with 5.9×10⁵ specific binding sites per cell. A similar apparentK_(a) was calculated for COS pCAV-18 cells at 1.5×10⁹ M⁻¹ but receptornumber expressed at the cell surface was 4.2×10⁴. Equilibrium bindingstudies performed on COS cells transfected with IL-4R DNA clonesisolated from the 7B9 cell library also showed high affinity binding ofthe receptor to IL-4. Specifically, studies using COS cells transfectedwith pCAV-7B9-2 demonstrated that the full length murine IL-4 receptorbound ¹²⁵ I-IL-4 with an apparent K_(a) of about 1.4×10¹⁰ M⁻¹ with4.5×10⁴ specific binding sites per cell. The apparent K_(a) of CAV-7B9-4IL-4 R was calculated to be about 1.7×10⁹ M⁻¹. Although absolute valuesfor Ka and binding sites per cell varied between transfections, thebinding affinities were generally similar (1×10⁹ -1×10¹⁰ M⁻¹) andmatched well with previously published affinity constants for IL-4binding.

Inhibition of ¹²⁵ I-mlL-4 binding to CTLL cells by conditioned mediafrom COS cells transfected with plasmid pCAV, pCAV-18, or pCAV-7B9-4 wasused to determine if these cDNAs encoded functional soluble receptormolecules. Approximately 1.5 μl of COS pCAV-18 conditioned media in afinal assay volume of 150 μl gives approximately 50% inhibition of ¹²⁵I-IL-4 binding to the IL-4 receptor on CTLL cells. ¹²⁵ I-IL-4 receptorcompeting activity is not detected in control pCAV transfected COSsupernatants. From quantitative analysis of the dilution of pCAV-18supernatant required to inhibit ¹²⁵ -IL-4 binding by 50%, it isestimated that approximately 60-100 ng/ml of soluble IL-4 receptor hasbeen secreted by COS cells when harvested three days after transfection.Similar results were obtained utilizing supematants from COS cellstransfected with pCAV-7B4.

Conditioned medium from COS cells transfected with pCAV-18 or pCAV-7B9-4(see Example 8) and grown in DMEM containing 3% FBS was harvested threedays after transfection. Supernatants were centrifuged at 3,000 cpm for10 minutes, and frozen until needed. Two hundred ml of conditioned mediawas loaded onto a column containing 4 ml of muIL-4 Affigel prepared asdescribed above. The column was washed extensively with PBS and IL-4receptor eluted with 0.1M glycine, 0.15 M NaCI pH 3.0. Immedatelyfollowing elution, samples were neutralized with 80 μl of 1M Hepes pH7.4. Samples were tested for their ability to inhibit binding of ¹²⁵I-muIL-4 to CTLL cells as set forth in Example 1B. Additionally sampleswere tested for purity by analysis on SDS-PAGE and silver staining aspreviously described. Alternative methods for testing functional solublereceptor activity or IL-4 binding inhibition include solid-phase bindingassays, as described in Example 1C, or other similar cell free assayswhich may utilize either radio iodinated or calorimetrically developedIL4 binding, such as RIA or ELISA. The protein analyzed by SOS-PAGEunder reducing conditions has a molecular weight of approximately37,500, and appears approximately 90% pure by silver stain analysis ofgels.

Purified recombinant soluble murine IL-4 receptor protein may also betested for its ability to inhibit IL-4 induced ³ H-thymidineincorporation in CTLL cells. Pursuant to such methods, soluble IL-4receptor has been found to block IL-4 stimulated proliferation, but doesnot affect IL-2 driven mitogenic response.

Molecular weight estimates were performed on mIL-4 receptor clonestransfected into COS cells. Utilizing M2 monoclonal antibody preparedagainst murine CTLL 19.4 cells (see Example 13), IL-4 receptor isimmunoprecipitated from COS cells transfected with CAV-16, CAV-7B9-2 andCAV7B9-4 and labeled with ³⁵ S-cysteine and ³⁵ S-methionine. Cellassociated receptor from CAV-7B9-4 shows molecular weight heterogeneityranging from 32-39 kDa. Secreted CAV-7B9-4 receptor has molecular weightbetween 36 and 41 kDa. Cell associated receptor from CAV-16 transfectedCOS cells is about 40-41 kDa. This is significantly smaller thanmolecular weight estimations from crosslinking studies described by Parket al., J. Exp. Med. 166:476, 1987; J. Cell. Biol., Suppl. 12A, 1988.Immunoprecipitation of COS CAV-7B9-2 cell-associated receptor showed amolecular weight of 130-140 kDa, similar to the estimates of Park etal., J. Cell. Biol. SuppI. 12A, 1988, estimated to be the full lengthIL-4 receptor. Similar molecular weight estimates of cell-associatedCAV-16 and CAV7B9-2 IL-4 receptor have also been made based oncross-linking ¹²⁵ IL-4 to COS cells transfected with these cDNAs.Heterogeneity of molecular weight of the individual clones can bepartially attributed to glycosylation. This data, together with DNAsequence analysis, suggests that the 7B9-2 cDNA encodes the full lengthcell-surface IL-4 receptor, whereas both 7B9-4 and clone 18 representsoluble forms of murine IL-4 receptor.

Receptor characterization studies were also done on COS cellstransfected with hIL-4R containing expression plasmids. The two chimerichuman IL-4R molecules A5 and B4 (defined in Example 11) were transfectedinto COS cells and equilibrium binding studies undertaken. The COSmonkey cell itself has receptors capable of binding hlL-4; therefore thebinding calculations performed on COS cells transfected with andoverexpressing hlL-4R cDNAs represent background binding from endogenousmonkey IL-4 R molecules subtracted from the total binding. COS cellstransfected with hIL-4R A5 had 5.3×10⁴ hIL-4 binding sites with acalculated K_(a) of 3.48×10⁹ M⁻¹. Similarly, the hIL-4 R B4 expressed inCOS cells bound ¹²⁵ I-hlL-4 with an affinity of 3.94×10⁹ M⁻¹ exhibiting3.2×10⁴ receptors per cell.

Molecular weight estimates of human ILAR expressed in COS cells werealso performed. COS cells transfected with clones AS or B4 in pCAV/NOTwere labeled with ³⁵ cysteine/methionine and lysed. Human IL-4R wasaffinity purified from the resulting lysates with hlL-4-coupled Affigel®(as described in Example 4). The hIL-4 R A5 and B4 eluted from thisaffinity support migrated at about 140,000 daltons on SDS-PAGE, agreeingwell with previous estimates of hIL-4R molecular weight by cross-linking(Park et al., J. Exp. Med. 166:476,1987), as well as with estimates offull-length mIL-4R presented here.

Because no soluble human IL-4R cDNA has thus far been found occurringnaturally, as was the case for the murine receptor (clones 18 and7B9-4), a truncated form was constructed as described in Example 11.Following expression in COS cells, supernatants were harvested threedays after transfection with soluble hIL-4 R-1 1 and soluble hIL-4 R-5and tested for inhibition of ¹²⁵ IhIL-4 binding to the human B cell lineRaji. Supernatants from two of the soluble hIL-4R-11 and one of thesoluble hIL-4R-5 transfected plates contained 29-149 ng/ml of IL-4 Rcompeting activity into the medium. In addition, the truncated proteincould be detected in ³⁵ S-methionine/cysteine-labeled COS celltransfectants by affinity purification on hlL-4-coupled Affigel® asapproximately a 44 kDa protein by SDS-PAGE.

Example 13 Preparation of monoclonal antibodies to IL-4R

Preparations of purified recombinant IL-4 receptor, for example, humanor murine IL-4 receptor, transfected COS cells expressing high levels ofIL-4 receptor or CTLL 19.4 cells are employed to generate monoclonalantibodies against IL-4 receptor using conventional techniques, such asthose disclosed in U.S. Pat. No. 4,411,993. Such antibodies are likelyto be useful in interfering with IL-4 binding to IL-4 receptors, forexample, in ameliorating toxic or other undesired effects of IL-4.

To immunize rats, IL-4 receptor bearing CTLL 19.4 cells were used asimmunogen emulsified in complete Freund's adjuvant and injected inamounts ranging from 10-100 μl subcutaneously into Lewis rats. Threeweeks later, the immunized animals were boosted with additionalimmunogen emulsified in incomplete Freund's adjuvant and boosted everythree weeks thereafter. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot-blotassay, ELISA (enzyme-linked immunosorbent assay), or inhibition ofbinding of ¹²⁵ I-IL-4 to extracts of CTLL cells (as described in Example1). Other assay procedures are also suitable. Following detection of anappropriate antibody filter, positive animals were given a finalintravenous injection of antigen in saline. Three to four days later,the animals were sacrificed, splenocytes harvested, and fused to themurine myeloma cell line AG8653. Hybridoma cell lines generated by thisprocedure were plated in multiple microtiter plates in a HAT selectivemedium (hypoxanthine, aminopterin, and thymidine) to inhibitproliferation of nonused cells, myeloma hybrids, and spleen cellhybrids.

Hybridoma clones thus generated were screened for reactivity with IL-4receptor. Initial screening of hybridoma supematants utilized anantibody capture and binding of partially purified ¹²⁵ I-mIL-4 receptor.Two of over 400 hybridomas screened were positive by this method. Thesetwo monoclonal antibodies, M1 and M2, were tested by a modified antibodycapture to detect blocking antibody. Only M1 was able to inhibit ¹²⁵I-rmIL-4 binding to intact CTLL cells. Both antibodies are capable ofimmunoprecipitating native mlL-4R protein from CTLL cells or COS-7 cellstransfected with IL-4R clones labelled with ³⁵ S-cysteinelmethionine. M1and M2 were then injected into the peritoneal cavities of nude mice toproduce ascites containing high concentrations (>1 mg/ml) of anti-IL-4Rmonoclonal antibody. The resulting monoclonal antibody was purified byammonium sulfate precipitation followed by gel exclusion chromatography,and/or affinity chromatography based on binding of antibody to ProteinG.

A series of experiments (Examples 14-19) was conducted to show thatsIL-4 R inhibits IL-4 mediated B cell growth, differentiation andfunction in vitro. Each of these experiments utilized soluble IL-4Rproduced as described in Example 8C. Example 14 shows that sIL-4Rinhibits the proliferation of stimulated B cells. Examples 15,16 and 17show, respectively, that sIL-4 R inhibits IL-4 dependent B celldifferentiation as measured by induction of IgG1 and IgE secretion byLPS activated B cells, down regulation of IgG3 secretion by LPSactivated B cells, and increased la and FcεR (CD23) expression,respectively. In the following experiments, the activity of sIL-4R iscompared with sIL-1R to show that the inhibitory effects of the solublereceptors are specific in that sIL-4R has no effect on IL-1 induced Bcell activity and sIL-1R has no effect on IL-4 activity, thusdemonstrating two independent pathways of B cell activation directed byIL-1 and IL-4.

Example 14 Inhibition of IL-4 Binding to B cells in vitro by SolubleIL-4R

Untreated B cells express low, but detectable levels of IL-4 receptors.Upon stimulation with the B cell mitogen LPS, these cells show enhancedcell surface IL-4 receptor expression. The following experiments wereconducted to show that sIL-4R inhibits radiolabeled IL-4 binding to LPSactivated B lymphocytes.

B lymphocytes were first purified from spleens of 8 to 12 week-oldC57BU6 mice (Jackson Laboratory, Bar Harbor, Me., and Simonson, Gilroy,Calif.) as described by Grabstein, et al., J. Exp. Med. 163:1405, 1986.Briefly, murine splenocytes were depleted of T cells by incubation in acocktail containing T24 rat anti-Thy 1 mAb (Dennert et al., J. Immunol131:2445 (1983), GK1.5 rat anti-mouse L3T4 mAb (Dialynas et al., Cell.Immunol. 53:350, (1980), rabbit anti-mouse thymocyte serum (absorbedwith C57BL/6 liver and bone marrow), and rabbit complement (Pel-FreezeBiologicals, Rogers, Ark.). Cells were then passed over Sephadex G-10(Pharmacia Uppsala, Sweden) to remove adherent cells. B lymphocytes werepositively selected by panning on petri dishes coated with affinitypurified goat anti-mouse igM (Organon Teknika Corp., West Chester, Pa.).The resultant preparations were >98% B cells as determined by flowcytometry.

The purified B cells were then cultured in RPMI 1640 supplemented with5% fetal calf serum (Hazelton), sodium pyruvate (1 mM), nonessentialamino acids (0.1 mM), penicillin (100 U/ml), streptomycin (100 ug/ml),L-glutanine (2 mM), and 2-mercaptoethanol (50 uM), as well as Salmonellatyphimuium LPS (10 ug/ml; Difco Laboratories, Detroit, Mich.) to produceactivated B cells.

Vuffan rIL-1β was produced in Eschetichia coli and purified tohomogeneity as described by Kronheim et al., Bio/Technology 4:1708,1986. Recombinant murine IL-4 was produced in yeast, purified tohornogeneity, and radiolabeled as described by Mosley et al., Cell59:355, 1989, and Park et al., Proc. Natl. Acad. Sci. USA 84:1 669,1987.

Inhibition assays were performed by first incubating variousconcentrations of unlabeled cytokines (IL-1 or IL-4), soluble receptors(sIL-1R or sIL-4R), monoclonal antibody (11B11, a rat IgGI anti-murineIL-4 antibody produced as described by Ohara et al., Nature 315:333,1985) or medium control with 50 ul ¹²⁵ l-labeled IL-4 (1.65×10⁻¹⁰ M) forthirty minutes in 10% CO₂ at 37° C. in binding medium (RPMI/2.5% BSA/0.2%sodium azidel 0.2M Hepes, pH 7.4). To these 2×10⁶ murine B cellswere added in 50 ul of binding medium for 30 min at 37° C. Cells werethen separated by the phthalate oil method as described by Dower et al.,J. Immunol. 132:751, 1984. Percent specific inhibition was calculatedusing incubation of ¹²⁵ I-IL-4 with excess unlabeled IL-4 (4×10⁻⁹ M) aspositive control and 50 ul of binding medium as negative control. Eachassay was performed with 3-fold dilutions in duplicate of eachcompetitor compound through binding medium, and incubations carried outin 96-well round bottom plates (Linbro, Hamden, Conn.).

Results of the inhibition assays indicate that IL-4 binding wasinhibited by unlabeled sIL-4R, unlabeled IL-4, and 11B11, an anti-IL-4specific mAb. The blocking effect was cytokine specific, andcross-competition between IL-4 and either 11B11 or sIL-4R generatedsimilar inhibition constants as shown in Table A below. No competitionof IL-4 binding was detected by IL-1 or sIL-1R.

                  TABLE A    ______________________________________    Inhibition of Radiolabeled IL-4 Binding to LPS Blasts    Inhibitor        Inhibition Constant (M.sup.-1)    ______________________________________    IL-4             4.9 × 10.sup.10    sIL-4R           4.8 × 10.sup.9    11B11            7.9 × 10.sup.9    IL-1             No Inhibition    sIL-1R           No Inhibition    ______________________________________

Example 15 Inhibition of Lymphokine Induced B Cell Proliferation invitro by sIL-4R

Murine B cell proliferation is stimulated by treatment ofanti-immunoglobulin and either IL-1 (Howard et al., J. Exp. Med.157:1529, 1983; Booth et al., J. ImmunoL 33:1346, 1984) or IL-4(Grabstein et al., J. Mol. Cell. Immunol 2:199,1986; Howard et al, J.Exp. Med. 155:914,1982). The ability of sIL-1R and sIL-4R to inhibitthese B cell mitogenic responses was tested in a B cell proliferationassay as follows.

B cells were purified and cultured as described in Example 14 above. Inorder to determine the effect of various doses of inhibitors on B cellproliferation, the purified B cells were seeded at 1×10⁵ cells/well in96-well flat-bottom tissue culture plates (Costar) in the presence ofaffinity purified goat anti-mouse IgM (2.5 ug/ml; Zymed Laboratories,Inc., So. San Francisco, Calif.) and various concentrations of IL-4(panel A) or IL-1 (panel B), either alone (∘) or in the presence of 1000ng/ml sIL-4R (□), 1000 ng/ml sIL-1R (▪), or 555 ng/ml 11B11 () asinhibitors. After 2 days, cultures received 2 uCi/well of ³ H!thymidine(25 Ci/mmol; Amersham, Arlington Heights., Ill.) for 16 hours, and werethen harvested onto glass fiber filters. Incorporation of radioactivitywas measured by liquid scintillation spectrophotometry. Tritiatedthymidine incorporation for triplicate wells was determined for thefinal 16 hours of a three day culture period. Results are presented asmean cpm±SEM.

In order to determine the effect of various doses of inhibitor on theinhibition of B cell proliferation by cytokines, purified B cells wereseeded at 1×10⁵ cells/well in 96-well flat-bottom tissue culture plates(Costar) in the presence of affinity purified goat anti-mouse IgM (25μg/ml; Zymed Laboratories, Inc., So. San Francisco, Calif.) and fixedconcentrations of 10 (), 1 (□), 0.1 (∘), or 0 (Δ) ng/ml of IL-4 (panelsA-C) or IL-1 (panels D-F). Culture wells also included three-folddilutions of sIL-4R (panels A, D), 11B11 (panels B,E), or sIL-1R (panelsC,F). After 2 days, cultures received 2 uCi/well of ³ H!thymidine (25Ci/mmol; Amersham, Arlington Heights., Ill.) for 16 hours, and were thenharvested onto glass fiber filters. Incorporation of radioactivity wasmeasured by liquid scintillation spectrophotometry as indicated above.Tritiated thymidine incorporation for triplicate wells was determinedfor the final 15 hours of a three day culture period. Results arepresented as mean cpm±SEM.

FIGS. 6 and 7 show that sIL-4R and sIL-1R inhibitory activity was dosedependent and specific for the respective ligands. The inhibitoryeffects of the sIL-4R and 11B11 were virtually equivalent on a molarbasis, with half-maximal inhibition of IL-4-induced proliferationrequiring a 100-200 fold molar excess of either inhibitor. Half-maximalinhibition of IL-1 activity was achieved with a 300-400 fold molarexcess of sIL-1R.

Example 16 Inhibition of IL-4 Induced Immunoglobulin Secretion In Vitroby sIL-4R

IL-4 augments LPS-induced secretion of IgGi and IgE and inhibits IgG3production, possibly by a mechanism involving class switching from oneisotype of an antibody to another isotype. The ability of sIL-4R toinhibit IL-4 induced class switching in LPS-stimulated B cells wastested in the following assay that measures immunoglobulin secretionfrom LPS treated B cells.

B cells were purified and cultured as described in Example 14 above. Inorder to determine the effect of various doses of IL-4 on IgGI, IgE andIgG3 secretion, the purified B cells (1×10⁵ cells/well) were grown in96-well flat bottom plates in the presence of Salmonella typhimudum LPS(Difco Laboratories, Detroit, Mich.) and threefold dilutions of IL-4with sIL-4R (□), sIL-1R (▪) or 11B11 (), each at 555 ng/ml or mediumcontrol (A) (see FIG. 8). Six days after initiation of culture, cellswere pelleted by centrifugation at 750×g and culture supernatant fluidswere harvested.

Immunoglobulin (IgG1, IgG3, and IgE) levels were determined by anisotype specific sandwich ELISA technique as follows. 96-wellflat-bottom Linbro plates (Flow Laboratories, Inc., McLean, Va.) werecoated overnight with the appropriate (see below) first step isotypespecific antibody (100 ul) and washed. This and all subsequent washingsteps were done with phosphate buffered saline containing 0.05% Tween20, 6 rinses per cycle. Nonspecific sites were blocked by incubation forone hour with 150 ul of 5% nonfat dry milk. Test material (100 ul),either culture supernatant or isotype standard curve solutions (allsample and antibody dilutions in PBS/ 3% BSA), was added to each well,incubated for 1 hour, then washed. 100 ul of the appropriate (see below)horseradish peroxidase-conjugated second step antibody was added andplates were incubated for 1 hour and washed. The presence ofperoxidase-conjugated antibody was determined by using the TMB Microwellperoxidase substrate system (Kirkegaard & Perry Laboratories, Inc.,Gaithersburg, Md.). Plates were read on a Dynatech ELISA reader.Immunoglobulin concentrations in test samples were determined bycomparing triplicate test values with isotype control standard curves,using the DeltaSoft 1.8 ELISA analysis program for the Macintosh(Biometallics, Inc., Princeton, N.J.).

For the IgG1 and IgG3 assays, unconjugated and horseradishperoxidase-conjugated affinity purified goat anti-mouse isotype specificreagents (Southern Biotechnology Associates, Inc., Birmingham, Ala.)were used as plate coating and second step reagents, respectively.Standard curves for IgG1 and IgG3 were run with isotype matched murinemyeloma proteins (Southern). For the IgE assay, the EM95 IgG2aanti-mouse IgE mAb (Banlyash et al., Eur. J. ImmunoL 14:797, 1984)(provided by Dr. Fred Finkelman, Uniformed Services, Bethesda, Md.) wasused as plate coating step reagent and biotinylated rat anti-mouse IgE(Bioproducts for Science, Inc., Indianapolis, Ind.) was used as secondstep reagent, and horse radish peroxidase-conjugated streptavidin(Zymed) was used in the third step. Standard curves were establishedwith a murine anti-dinitrophenol specific IgE myeloma antibody (ATCC No.TIB 141). All three ELISA assays were determined to be specific basedupon cross-reactivity experiments using all individual murine antibodyisotypes as controls.

The effect of various doses of inhibitor on the inhibition of IgG1, IgG3and IgE secretion is shown in FIG. 9. In this experiment, purified Bcells (1×10⁵ cells/well) were grown in 96-well flat bottom plates in thepresence of Salmonella typhimunum LPS (Difco Laboratories, Detroit,Mich.) and IL-4 (30 ng/ml for IgE; 3 ng/ml for IgG1 and IgG3) in thepresence of three-fold dilutions of sIL-4R (□), sIL-1R 1R (M) or 11B11(). Six days after initiation of culture, cells were pelleted bycentrifugation at 750×g and culture supernatant fluids were harvested.The supematants were analyzed for IgG1, IgG3 and IgE secretion using theisotype specific sandwich ELISA technique described above.

FIG. 8 (panels A and B) shows that IgG1 and IgE secretion from LPStreated B cells was induced by IL-4 and that these activities wereinhibited by both the sIL-4R as well as 11B11. In contrast, panel Cshows that IgG3 secretion was induced by LPS directly in the absence ofexogenous cytokines. When IL-4 was present at concentrations of 10 ng/mlor less, LPS induced IgG3 secretion was ablated. sIL-4 R blocked thisinhibitory effect of IL-4, shifting the IL-4 dose response curve andeffectively permitting induction of IgG3 secretion in the presence ofotherwise inhibitory doses of IL-4.

FIG. 9 shows that the inhibition of IL-4 induced class switching bysIL-4R was dose dependent: with increasing concentrations of inhibitors,progressively lower levels of IgG1 and IgE and progressively higherlevels of IgG3 were secreted. sIL-1R had no such effect, even at thehigher concentrations (>1 ug/ml).

Example 17 Inhibition of IL-4 Induced Cell Surface Antigen Egression onB Cells In Vitro by sIL-4R

IL-4 induces increased expression of various cell surface antigens onresting murine B cells. In order to determine the effect of sIL-4R onthe inhibition of cell surface antigen expression, fluorescent-labeledantibodies to two specific cell surface antigens, MHC class 11 (1a)antigens and FeεR (CD23), were used to measure the level of antigenexpressed on B cells as follows.

Inhibition of MHC dass 11 (1a) Antigens. Purified B cells (5×10⁵cells/ml) were cultured for 16 h in 24-well plates (Costar) with orwithout IL-4 (0.1 ng/ml) in the presence of sIL-4 R, 11B11 or sIL-1Reach at 500 n/ml or medium control with or without inhibitors. Cellswere washed and preincubated for 20 min on ice with the rat IgG2banti-murine FcγR mAb 2.4G2 (Unkeless, et al., J. Exp. Med. 150.580,1979) to block IgG Fc receptors. Fluorescinated mAbs (25-9-17, a murineIgG2a anti-murine I-A^(b) antibody, described by Ozato et al., J.Immunol 126:317, 1981; or control murine IgG1) were added directly andcells were incubated for 30 min at 4° C. and washed. The antibodydiluent and wash solution was PBS/1% fetal calf serum/.01% NaN₃. Stainedcells were analyzed on a FACScan flow cytometer (BecktonDickinson, SanJose, Calif.) using a logarithmic fluorescence intensity scale.

FIG. 10 shows that in medium control (panel A) la expression in B cellsis significantly greater in the presence of IL-4 (dashed line) thanwithout IL-4 (solid line). Addition of sIL-4R (panel B) or 11b11 (panelC) at the onset of culture returned la expression to constitutivelevels, whereas addition of sIL-1R (panel D) had no effect. The resultsshown in FIG. 10 are representative of 3 separate experiments.

Inhibition of FcεR (CD23). IL-4 also induces expression of CD₂₃ onmurine and human B cells. B cells were cultured without or without IL-4in the presence of sIL-4R, 11B11, sIL-1R or medium control as describedabove. Cells were stained with FITC-labeled anti-CD23 antibody (B3B4, arat IgG2a anti-murine FceR CD23!, described by Rao et al., J. Immunol.138:1845, 1987) as described above.

FIG. 11 shows that in medium control (panel A) CD23 expression in Bcells is significantly greater in the presence of IL-4 (dashed line)than without IL-4 (solid line). Addition of sIL-4R (panel B) or 11B11(panel C) returned cell surface CD23 expression to constitutive levels,whereas addition of sIL-1R (panel D) had no effect. As with laexpression, the sIL4R and 11B11 inhibitors did not diminish theconstitutive expression of CD23, indicating that the inhibition waslimited to the IL4 dependent increase. The results shown in FIG. 10 arealso representative of 3 separate experiments.

Examples 18 and 19 are experiments which show the effect of sIL-4R onthe inhibition of IgE responses in vivo. Example 18 shows that sIL-4Rinhibits an IgE response to a specific antigen. Example 19 shows thatadministration of sL-4R in doses ranging from 1-25 ug twice daily ondays -1, 0 and +1 do not inhibit an IgE response to a cocktail ofanti-IgD antibodies.

Example 18 Inhibition of IL-4 Dependent Antigen-Specific IgE Response ofB Cells by sIL-4R In Vivo

Animals immunized with the hapten-carrier conjugate TNP-KLH(trinitrophenol-keyhole limpet hemocyanin) in alum generate a stronganti-TNP IgE antibody response. In order to determine the effect ofsIL-4R administration on the IgE response, the following experiment wasconducted.

Balbic mice (3 mice/group, 7 groups) were immunized i.p. with 1 ug ofTNP-KLH in alum on day 0. On day 21, the mice were boosted with the sameamount of TNP-KLH, then bled 5 days later. Serum was then assayed byimmunoglobulin isotype-specdic ELISA for levels of both polyclonal andantigen-specific (TNP-specific) immunoglobulin. This secondary antibodyresponse is characterized by, although not restricted to, the generationof a sng IggE response, most of which is anti-TNP specific.

On days -1, 0, and +1 of the secondary immunization, mice were giventwice-daily injections of sIL-4 R in total daily doses of 25, 5, and 1ug/mouse. Thus, each mouse received a total of 75, 15, or 3 ug of sIL-4Rover the three day treatment period. Serum was prepared from eachanimal, and analyzed for polyclonal and anti-TNP IgE concentrations.

The results of these experiments, shown in FIG. 12, indicate thatuntreated mice (saline control), primary-immunized mice (1-no boost),and primed and boosted mice (1-2) displayed polyclonal IgE levels ofapproximately 1 ug/ml, 5 ug/ml, and 25 ug/ml, respectively. Treatment ofboosted mice with 11B11 anibJL4 antibody lowered IgE levels toapproximately 2 ug/ml. Treatment of boosted mice with sIL-4 R loweredIgE levels significantly, with the highest concentration of sIL-4R (25ug/hit) resulting in greater than 80% reduction in IgE. Lower doses ofsIL-4R inhibited the polyclonal IgE response, although lessdramatically. Thus, the sIL-4R acts as an inhibitor of anantigen-induced polyclonal IgE response.

FIG. 13 shows that priming of mice with TNP-KLH (1-no boost) resulted ina detectable anti-TNP response of the IgE isotype. Boosting with TNP-KLHcaused a significant increase in the anti-TNP IgE titre. Treatment ofprimed and boosted mice with 11B11 at the time of secondary immunizationdiminished the antigen-specific IgE levels to less than levels seen inprimed-only mice. The highest concentration of sIL-4R also dramaticallydecreased anti-TNP specific levels. Lower concentrations of sIL-4R hadno discernible effect upon the secondary IgE response to TNP-KLH.

Example 19 In vivo Inhibition of IL-4-Dependent Polyclonal IgE Responseof B Cells by sIL-4R

Animals immunized with a cocktail of monoclonal IgD antibodies specificfor murine IgD (allotype specific) generate a strong polyconal IgEantibody response. One likely mechanism of anti-IgD action involvescrosslinking of surface immunoglobulin on B cells, internalization andprocessing of the anti-lgD, and presentation to helper T cells. TheIg-allotype specific T cells are thus triggered to provide signals(presumably cytokines) which induce immunoglobulin class switching andsecretion by B cells.

In order to determine the effect of sIL-4R administration on theIgD-induced polyclonal IgE response, the following experiment wasconducted. BALB/cmice (3 mice/group) immunized i.v. with 800 ug ofanti-IgD were treated twice daily with three different doses of sIL-4R(12.5, 2.5, or 0.5 ug/injection) on days -1, 0, and +1. Mice were bledon day 9 and serum IgE levels were determined. FIG. 14 shows thatanti-IgD treatment (MSA control) caused large increases in levels ofsecreted IgE when compared with unimmunized controls (saline control).This effect was blocked by anti-IL-4 (11B11) administration, but not byany of the doses of sIL-4R. Anti-IgD treatment also caused largeincreases in IgG1, IgG2a, and IgG3. Whereas sIL-4R administration had noeffect upon these isotypes, 11B11 administration resulted in increasedIgG2a and IgG3 secretion.

The failure of sIL-4R to inhibit the IgE stimulatory effect of anti-IgDmay be due to the fact that the inhibitor must be present for longerthan ore day after anti-IgD treatment, that the 11B11 antibody has alonger serum half-life than the sIL-4R, or that higher doses of sIL4Rare required.

Example 20 Use of Soluble IL-4 Receptor to Inhibit ContactHypersensitivity Responses to DNFB

The effect of soluble IL-4 receptor (sIL-4R) and soluble IL-1 receptor(sIL-1R) on contact hypersensitivity (CRS) responses were evaluated in amurine system using 2,4-dinitrofluorobenzene (DNFB) as the contactsensitizer. Groups of female BALB/c mice (5 mice per group) were treatedwith either sIL-4R or sIL-1R on either days -1 through +1, days 4 and 5or days -1 through 5 with 500 ng, b.i.d. via intraperitoneal injection.Control mice were treated with equivalent doses of the carrier solutioncontaining mouse serum albumin (MSA). Epicutaneous sensitization withDNFB was performed by application of 25 ul of a solution of 0.5% DNFB in4:1 mixture of acetone:olive oil to the shaved backs of mice on day 0.Negative control mice were not sensitized to DNFB. CHS responses wereelicited by challenging all groups of mice on day 5 by application of 10ul of the DNFB solution to the right rear footpads of the mice in eachof the treatment groups. The extent of CHS induction was determined bymeasuring the difference in thickness (in units of 10⁻² mm) between thechallenged right and unchallenged left rear footpads (as measured with adial micrometer) 24 hours later. FIGS. 15 shows the results of theseexperiments. The data are presented as mean footpad swelling ±SEM.

As shown in FIG. 15, mice treated with sIL-1R developed CHS responsesthat were not significantly different from control mice treated with MSAregardless of the treatment regimen used. Mice treated with sIL-4R ondays -1 through 1 developed CHS responses that were not significantlydifferent from the MSA control group. Although there appeared to be aslight increase in the CHS responses to DNFB induced in mice treatedwith sIL-4R on days 4 and 5, inspection of the data indicated that thiswas primarily due to the enhanced response of only one out of five ofthe mice in the group. No significant effect of treatment (eitherenhancement or inhibition) is observed if the response of this singlemouse is considered to be an outlier and is not considered in theevaluation of the data (FIG. 5) However, mice treated with sIL-4R duringthe entire period (days -1 through 5) were significantly (p<0.001)inhibited relative to MSA-treated control mice, and not significantlydifferent from mice which had not been sensitized, but only challenged,with DNFB. This data thus indicates that sIL-4R is effective ininhibiting contact hypersensitivity responses to DNFB.

Example 21 Use of Soluble IL-4 Receptor to Inhibit Delayed-TypeHypersensitivity Responses to SRBC

The effect of soluble IL-4 receptor (sIL-4R) and soluble IL-1 receptor(sIL-1R) on delayed-type hypersensitivity (DTH) responses to sheep redblood cells (SRBC) were evaluated in a murine system as described byKitamura, J. Immunol Meth. 39:277, 1980. Three groups of female BALB/cmice (4 mice per group) were sensitized on day 0 by i.v. injection of2×10⁵ SRBC. A fourth group of negative control mice were not sensitizedto SRBC. DTH responses were elicited in the sensitized mice bychallenging the mice on day 4 with 1×10⁸ SRBC via intracutaneousinjection in the right rear footpads of the mice and 100 ul normalsaline in the contralateral footpad as a control. The mice were treatedwith either 0.1 ug or 1.0 ug of sIL-R in 100 ul MSA via intraperitonealinjection, on the day of challenge and the day of immunization. Controlmice were treated with equivalent doses of the carrier solution (100 ul)containing mouse serum albumin (MSA). The fourth group of negativecontrol mice not sensitized to SRBC were treated with 100 ul of MSA. Theextent of the DTH response induced was determined by excising thefootpads at the tarsus and measuring the difference in weight betweenSRBC challenged and saline challenged footpads. FIG. 16 shows theresults of these experiments. The data are presented as mean footpadswelling ±SEM.

As shown in FIG. 16, the DTH response was almost totally blocked byintraperitoneal injection with 1 ug sIL-4R on the day of challenge andthe day of immunization. Treatment with 0.1 ug sI-4R was less effective.Treatment with 0.2 ug or 2 ug of sIL-1R inhibited the response (notshown). These data thus indicate that sIL-4R is effective in inhibitingdelayed-type hypersensitivity responses.

Example 22 Use of Soluble IL-4R to Suppress Immune Response toAlloantigen in vivo

Experiments were conducted to show that systemic administration ofsIL-4R suppresses a localized, T cell-dependent, immune response toalloantigen presented by allogeneic cells. The response to allogeneiccells in vivo was quantified using the popliteal lymph node enlargementassay described by Twist et al., Transplantation 15: 182, 1973, which isused as a measure of allograft transplant immunity (see Grebe et al.,Adv. Immunol. 22:119, 1976). In this assay mice are injected in thefootpad with irradiated, allogeneic spleen cells. The mice are theninjected in the contralateral footpad with irradiated, syngeneic spleencells. An alloreactive response (marked by proliferation of lymphocytesand inflammation) occurs in the footpad receiving the allgeneic cells,which can be measured by determining the increase in size and weight ofthe popliteal lymph node draining the site of antigen depositionrelative to controls or by an increase in cellularity.

Specific pathogen free 8-12 week old BALB/c (H-2^(d)) and C57BL/6(H-2^(b)) mice (Jackson Laboratory, Bar Harbor, ME) were used in thisexperiment. 9 BALB/c Mice were divided into 3 groups, each having 3mice. Each group of mice received a different mode of treatment asindicated below in Tables B. On day 0 the left footpads of all mice wereinjected intracutaneously with 10⁷ irradiated (2500R), allogeneic spleencells from C57BL/6 mice in 50 ul of RPMI-1640 (Gibco) as antigen and theright contralateral footpads of the same mice were injected with 10⁷irradiated (2500R), syngeneic spleen cells from BALE/c mice. All dosesof soluble mutine IL-4 receptor (sIL-4R) were diluted in phosphatebuffered saline (PBS). On days -1, 0 and +1 three mice were injected(intravenously on days -1 and 0, and subcutaneously on day +1) with 100ng of purified smuIL-4R, three mice were injected intravenously with 1ug of smuIL-4R, three mice were injected with 2 ug of smuIL-4R and threemice were injected with MSA (control).

Seven days after antigen administration, the mice were sacrificed andthe popliteal lymph nodes (PLN) were removed from the right and leftpopliteal fossa by surgical dissection. Lymph nodes were weighed and theresults expressed as the difference (Δ) in weight (mg) of the lymph nodedraining the site of aflgeneic cell injection and the weight of the nodedraining the syngeneic cell injection site (Table B; FIG. 17). The meandifference in weight of the lymph nodes from the sites of allogeneic andsyngeneic spleen cells was approximately 2.5 mg for the mice treatedwith MSA, 1 mg for the mice treated with 100 ng of sIL-4R, and 0.5 mgfor mice treated with 1 ug sIL-4R. No detectable difference in weight oflymph nodes was ascertainable for the mice treated with 2 ug sIL-4R.Lymph nodes draining the syngeneic cell injection site weighedapproximately 1 mg, regardless of whether they were obtained from micetreated with MSA or smuIL-4R, and did not differ significantly in weightfrom nodes obtained from mice given no cell injection. Values forstatistical significance were calculated using the two-tailed Student'st-test. Thus, IL-4R significantly (p<0.1 in all groups, using atwo-tailed T test) suppressed the in viva lymphoproliferative responsein a dose dependent fashion relative to control mice.

                  TABLE B    ______________________________________    Effect of smuIL-4R Administration on Proliferation of Lymph Node Cells    Treatment   Weight (mg) of Lymph Node    Group       Allogeneic Syngeneic Δ    ______________________________________    MSA         3.9 ± 0.06                           1.27 ± 0.07                                     2.63 ± 0.1    100 ng smuIL-4R                2.3 ± 0.03                            1.3 ± 0.03                                      1.0 ± 0.06    1 ug smuIL-4R                2.1 ± 0.9                           1.9 ± 0.3                                     0.23 ± 0.6    2 ug smuIL-4R                1.6 ± 0.3                           1.5 ± 0.1                                     0.0 ± 0.4    ______________________________________

Table B shows that systemic administration of sIL-4R for 3 daysbeginning on day -1 relative to alloantigenic challenge resulted in adramatic decrease in the size of lymph nodes, indicating that thelymphoproliferative response is inhibited. The effect was dose dependentand, in some cases, the response was virtually eliminated.

Example 23 Use of Soluble IL-4 Receptor to Suppress Allograft Rejection

Soluble murine IL-4 receptor also suppresses rejection of organ graftsin vivo. In order to demonstrate this, neonatal C57BL/6 (H-2^(b)) heartswere transplanted into the ear pinnae of adult BALB/c (H-2^(d))recipients utilizing the method of Fulmer et al., Am. J. Anat. 113.273,1963, modified as described by Trager et al., Transplantation47:587,1989, and Van Buren et al., Transplant. Proc. 15:2967, 1983.Survival of the transplanted hearts was assessed by visually inspectingthe grafts for pulsatile activity. Pulsatile activity was determined byexamining the ear-heart grafts of anesthetized recipients under adissecting microscope with soft reflected light beginning on day 5 or 6post transplant. The time of graft rejection was defined as the dayafter transplantation on which contractile activity ceases.

Recipient mice were divided into two groups, a primary treatment groupand a secondary treatment group. The primary treatment group were notexposed to antigen from C57BL/6 mice previous to being treated, whilethe secondary treatment group had been exposed to antigen. All mice weretransplanted on day 0 and injected with either smuIL-4R (1000 ng/day)plus MSA (mouse serum albumin, 100 ng) or with MSA alone on days 0through 2, i.p. The results of this experiment are reported below inTable C. The probability (p value) that the survival time for the grouptreated with smuIL-4R differs by chance alone from the group treatedwith MSA is less than 0.04 when analyzed by the Student's t-test for theprimary treatment group. The corresponding p values for secondarytreatment group are not significant.

                  TABLE C    ______________________________________    Effects of smuIL-4R Treatment on Nonvascularized Heterotopic    Cardiac Allograft Survival    Treatment                 Median Survival Time ±    Group       Survival Time (days)                              S. D.    ______________________________________    Primary Treatment    MSA (100 ng)                9, 10, 12, 14 11.3 ± 1.1    smuIL-4R (100 ng)                10, 10, 10, 12                              10.5 ± 0.5    SmuIL-4R (1000 ng)                12, 14, 14, 16, 17, 19                              15.3 ± 1.0    Secondary Treatment    MSA (100 ng)                8, 8, 8         8 ± 0.0    smuIL-4R (1000 ng)                8, 10, 12       10 ± 1.2    ______________________________________

Table C shows that heart allografts survived 9-14 days in individualcontrol mice treated with MSA. When primary allograft recipients weregiven 3 daily injections of 1000 ng smuIL-4R, graft survival wasprolonged. The median graft survival time in smulL-4R treated mice(14-19 days) was approximately four days longer than the median graftsurvival time of identical grafts in control mice. A subtle increase ingraft survival following secondary transplantation suggests that acuterejection episodes are influenced by smulL-4R administration as well.This data is evidence of the therapeutic potential of soluble human IL-4receptor in humans for the suppression of heart allograft rejection.

We claim:
 1. An isolated DNA sequence encoding a biologically activehuman IL-4 receptor (IL-4R), selected from the groups consisting of:(a)CDNA clones that encode a human IL-4R comprising the amino acid sequenceof amino acids 1-800 in FIGS. 4A-4C, (b) DNA sequences capable ofhybridization to the clones of (a) under moderately stringent conditionsand which encode biologically active human EL-4R; and (c) DNA sequenceswhich are degenerate as a result of the genetic code to the DNAsequences define in (a) or (b) and which encode biologically activehuman IL-4R.
 2. A purified polypeptide according to claim 1, whereinsaid IL-4R polypeptide is a soluble polypeptide comprising an amino acidsequence that is identical to the sequence presented as amino acids 1 to207 of FIG. 4A or deviating therefrom by no more than one amino aciddeletion, insertion or substitution.
 3. A pharmaceutical compositioncomprising a soluble polypeptide according to claim 2 in admixture witha suitable diluent, excipient, or carrier.
 4. A purified EL-4Rpolypeptide, comprising a fragment selected from the group consistingof:a) an immunogenic fragment of the murine protein of FIGS. 2A-2C andb) an immunogenic fragment of the human protein of FIGS. 4A-4C.
 5. Apurified polypeptide according to claim 4, wherein said fragment iscapable of binding IL-4.
 6. A pharmaceutical composition comprising asoluble polypeptide according to claim 5 in admixture with a suitablediluent, excipient, or carrier.
 7. A purified polypeptide comprising anamino acid sequence that is at least 80% identical to a sequenceselected from the group consisting of:a) amino acids 1 to 785 of FIGS.2A-2C; b) amino acids 1 to 800 of FIGS. 4A-4C; c) amino acids 1 to 208of FIG. 2A; and d) amino acids 1 to 207 of FIG. 4A: wherein saidpolypeptide is a naturally occurring polypeptide that is capable ofbinding IL-4.
 8. A purified polypeptide according to claim 7, whereincysteine residue(s) in said polypeptide have been deleted or replacedwith non-cysteine residue(s).
 9. A purified polypeptide, wherein saidpolypeptide is encoded by a DNA selected from the group consisting of:a)a DNA comprising the nucleotide sequence presented as nucleotides 1 to2355 of FIGS. 2A-2C: b) a DNA comprising the nucleotide sequencepresented as nucleotides 1 to 2400 of FIGS. 4A-4C: and c) a DNA thathybridizes under moderately stringent conditions to a DNA of (a) or (b);wherein said polypeptide is a naturally occurring polypeptide that iscapable of binding IL-4.
 10. A purified polypeptide that is a fragmentof a naturally occurring polypeptide according to claim 9, wherein saidfragment is capable of binding IL-4.
 11. An isolated polypeptideselected from the group consisting of:a) an immunogenic fragment of themurine protein of FIGS. 2A-2C; and b) an immunogenic fragment of thehuman protein of FIGS. 4A-4C.
 12. An isolated polypeptide capable ofbinding IL-4, selected from the group consisting of the murinepolypeptide of FIGS. 2A-2C and the human polypeptide of FIGS. 4A-4C,with the proviso that said polypeptide is devoid of a transmembraneregion.